Haemophilus outer membrane protein

ABSTRACT

Purified and isolated nucleic acid from specific strains of  Haemophilus influenzae  is provided which encodes at least a portion of the D15 outer membrane protein of Haemophilus. The nucleic acid is used to produce peptides, polypeptides and proteins free of contaminant associated with Haemophilus for purposes of diagnosis and medical treatment. Furthermore, the nucleic acid may be used in the diagnosis of Haemophilus infection. Antisera obtained following immunization with the nucleic acid D15 outer membrane protein or peptides also may be used for the purpose of diagnosis and medical treatment.

This is a division of Application Ser. No. 08/433,522 filed Sep. 12,1995, which is a National Phase filing from PCT/CA93/00501 filed Nov.23, 1993.

FIELD OF INVENTION

The present invention is related to the field of molecular genetics andis particularly concerned with the cloning of an outer membrane proteinD15 of Haemophilus.

BACKGROUND OF THE INVENTION

Haemophilus influenzae type b (Hib) is a major cause of bacterialmeningitis in children under the age of five years. Protectiveantibodies to the disease are induced by the capsular polysaccharide ofthe organism and a vaccine was developed that utilises the purifiedpolyribosyl ribitol phosphate (PRP) as the antigen. This vaccineprovides 90% protection in adults and in children over 24 months of age,but was ineffective in children under 24 months Zangwill et al 1993 (Thereferences are identified in a list of reference at the end of thisdisclosure). Like other polysaccharide antigens, PRP does not induce theproliferation of T-helper cells, and re-immunisation fails to eliciteither a booster response or an increase in memory cells. Conjugation ofthe PRP polysaccharide with protein carriers confers T-cell dependentcharacteristics to the vaccine and substantially enhances theimmunologic response to the PRP antigen. Currently, there are fourPRP-carrier conjugate vaccines available. These are vaccines based uponH. influenzae type b capsular polysaccharide conjugated to diphtheriatoxoid, tetanus toxoid, or Neisseria meningitidis outer membrane protein(reviewed in Zangwill et al 1993).

However, the current Haemophilus conjugate vaccines only protect againstmeningitis caused by Haemophilus influenzae type b. They do not protectagainst other invasive typeable strains (types a and c) and, moreimportantly, against non-typeable (NTHi) strains which are a commoncause of postpartum and neonatal sepsis, pneumonia and otitis media. Inthe United States alone, treatment of otitis media costs between 1 and 2billion dollars per year for antibiotics and surgical procedures, suchas tonsillectomies, adenoidectomies and insertion of tympanostomy tubes.To achieve universal protection against H. influenzae related diseasesin the 2 to 6 month age group and certain high risk groups, theprovision of conserved, cross-reactive non-capsular H. influenzaeimmunogens is desirable. Methods for inducing immunity against diseaseare constantly improving and there is presently a move to use subunitsand better defined materials as antigens. This is being undertaken tominimise or eliminate potential side-effects caused by certain nativeimmunogens, while preserving their immunogenicity to confer protectionagainst the disease. Therefore, it would be very attractive to develop auniversal vaccine against Haemophilus using cross-reactive outermembrane proteins, fragment, analogs, and/or peptides correspondingthereto as protective antigens. Such antigens may be incorporated intothe conventional H. influenzae type b conjugate vaccines as additionalimmunogens or used as autologous carriers for H. influenzae capsularpolysaccharides. A high molecular weight outer membrane protein D15found in non-typeable and type b stains of H. influenzae has beenidentified as a cross-reactive antigen (Thomas et al., 1990). D15appears to be cell surface-exposed in its natural state and exhibits amolecular mass of about 80 kDa as judged by SDS-PAGE analysis. It wouldbe desirable to provide the sequence of the DNA molecule that encodesthis D15 outer membrane protein and peptides corresponding to portionsthereof for diagnosis, immunization and the generation of diagnostic andimmunological reagents. The diseases caused by Haemophilus are seriousand improved methods for preventing, detecting and treating diseasessuch as otitis media, epiglottitis, pneumonia, and tracheobronchitis,are required.

The present invention is directed towards the provision of purified andisolated nucleic acid molecules comprising at least a portion coding fora D15 outer membrane protein of a species of Haemophilus. The nucleicacid molecules comprising at least a portion coding for D15 outermembrane protein are useful for the specific detection of strains ofHaemophilus, and for diagnosis of infection by Haemophilus. The purifiedand isolated nucleic acid molecules, such as DNA comprising at least aportion coding for D15 outer membrane protein, are also useful forexpression of the D15 gene by recombinant DNA means for providing, in aneconomical manner, purified and isolated D15 outer membrane protein.

The D15 outer membrane protein or fragments thereof or analogs thereofare useful immunogenic compositions for the preparation of vaccinesagainst diseases caused by Haemophilus, the diagnosis of infection byHaemophilus and as tools for the generation of immunological reagents.Mono- or polyclonal antisera (antibodies) raised against the D15 outermembrane protein produced in accordance with aspects of the presentinvention are useful for the diagnosis of infection by Haemophilus,specific detection of Haemophilus (in, for example, in vitro and in vivoassays) and for the treatment of diseases caused by infection byHaemophilus,

Peptides corresponding to portions of the D15 outer membrane protein oranalogs thereof are useful immunogenic compositions for the preparationof vaccines against disease caused by Haemophilus, the diagnosis ofinfection by Haemophilus and as tools for the generation ofimmunological reagents. Mono- or polyclonal antisera raised againstthese peptides, produced in accordance with aspects of the presentinvention, are useful for the diagnosis of infection by Haemophilus,specific detection of Haemophilus (in, for example, in vitro and in vivoassays) and for use in passive immunization as a treatment of diseasecaused by infection by Haemophilus.

In accordance with one aspect of the present invention, therefore, thereis provided a purified and isolated nucleic acid molecule, the moleculecomprising at least a portion coding for a D15 outer membrane protein.The nucleic acid molecule has a DNA sequence selected from:

(a) the DNA sequence set out in any one of FIGS. 1A to 1E (as describedbelow) or its complementary strand; and

(b) DNA sequences which hybridize under stringent conditions to the DNAsequences defined in (a). The DNA sequences defined in (b) preferablyhas at least 90% sequence identity with the sequences defined in (a).The DNA sequence defined in (b) particularly may comprise the consensussequence set forth in FIG. 1F (as described below).

In another aspect of the present invention, there is provided a purifiedand isolated D15 outer membrane protein or a portion thereof. The D15outer membrane protein may be a Haemophilus D15 outer membrane proteinand more particularly an H. influenzae D15 outer membrane protein andthe H. influenzae strain may be an H. influenzae type b strain, such asH. influenzae type b strains Ca or Eagan or MinnA or a non-typeable H.influenzae strain, such as PAK 12085 or SB33.

In an additional embodiment, the present invention also includes arecombinant plasmid adapted for transformation of a host, therecombinant plasmid comprising a plasmid vector into which has beeninserted a DNA segment comprising the purified and isolated DNA moleculeprovided herein. Such recombinant plasmid comprises a plasmid vectorinto which a DNA segment which comprises at least an 18 bp fragmentselected from the DNA molecules as recited above is inserted. Therecombinant plasmid may be plasmid DS-712-2-1 having ATCC accessionnumber 75604, deposited Nov. 4, 1993 and plasmid JB-1042-5-1 having ATCCaccession No. 75006, deposited Nov. 4, 1993.

The plasmids may be adapted for expression of the encoded D15 outermembrane protein in a host cell, which may be a heterologous orhomologous host, by incorporation into a recombinant vector, provided inaccordance with a further aspect of the invention. The recombinantvector may comprise at least a DNA segment comprising at least an 18 bpfragment selected from the DNA molecules as recited above and expressionmeans operatively coupled to the DNA segment for expression of the geneproduct encoded thereby in the host cell. The plasmid for expression ofthe encoded D15 outer membrane protein may be plasmid DS-880-1-2 havingATCC accession number 75605, deposited Nov. 4, 1993 being adapted forexpression at the D15 outer membrane protein in E. coli. The selectedDNA segment may encode a polypeptide of at least 6 residues and, inparticular, may be selected from those segments encoding a polypeptideof Table 2 (below). The DNA segment may further comprise a nucleic acidsequence encoding a leader sequence for export of the gene product fromthe host. The host for expression may be selected from, for example,Escherichia coli, Bacillus, Haemophilus, fungi, yeast or the baculovirusexpression system may be used.

Additional aspects of the invention include the protein encoded by theDNA molecule comprising at least a portion coding for the D15 outermembrane protein, fragment or a functional analog of such protein, theuse of the protein or analog in vaccination and diagnosis, and thegeneration of immunological reagents. The invention also includesantisera (antibodies) raised against the D15 outer membrane proteinencoded by the DNA molecule comprising at least a portion coding for aD15 outer membrane protein and purified peptides corresponding toportions of the D15 outer membrane protein and there are in passiveimmunization and treatment of diseases caused by Haemophilus.

According to another aspect of the invention, a purified and isolatedpeptide containing an amino acid sequence corresponding to the aminoacid sequence of at least a portion of the D15 outer membrane protein orvariant or mutant which retains immunogenicity. The peptide may beproduced by recombinant methods or peptide synthesis whereby thepurified peptide is free from contaminants associated with bacterianormally containing the D15 outer membrane protein. Such syntheticpeptides preferably have an amino acid sequence selected from thosepresented in Table 2.

In accordance with an additional aspect of the invention, an immunogeniccomposition is provided which comprises the D15 outer membrane protein,fragments thereof, functional analogs thereof, or peptides as recitedabove and a physiologically-acceptable carrier therefor. Suchimmunogenic composition is particularly formulated as a vaccine for invivo administration to protect against diseases caused by Haemophilus.For such purpose, the immunogenic composition may be formulated as amicroparticle preparation, capsule preparation or liposome preparation.In addition, such immunogenic composition may be provided in combinationwith a targeting molecule for delivery to specific cells of the immunesystem or to mucosal surfaces.

In accordance with a further aspect of the invention, there is provideda method for inducing protection against disease caused by Haemophilus,comprising the step of administering to a subject, including a mammal,such as a human, an effective amount of the immunogenic composition orthe nucleic acid molecule as recited above to provide protectiveimmunity against Haemophilus infection.

The present invention further includes a chimeric molecule comprising aD15 protein or peptide corresponding thereto as provided herein linkedto another polypeptide or protein or a polysaccharide. The linkedpolypeptide or protein may comprise a surface protein or peptidecorresponding thereto from a pathogenic bacteria, which may be the P1,P2 or P6 outer membrane protein of H. influenzae. The linkedpolysaccharide preferably comprise a PRP molecule from H. influenzae.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will be further understood from the followingdescription with reference to the drawings, in which:

FIGS. 1A-1 to 1A-8 show the nucleotide sequence of the D15 gene from H.influenzae type b Ca strain (SEQ ID NO: 1) and its deduced amino acidsequence (SEQ ID NO: 2);

FIGS. 1B-1 to 1B-8 show the nucleotide sequence of the D15 gene from H.influenzae type b Eagan strain (SEQ ID NO. 3) and its deduced amino acidsequence (SEQ ID NO: 4);

FIGS. 1C-1 to 1C-9 show the nucleotide sequence of the D15 gene from H.influenzae type b MinnA strain (SEQ ID NO. 5) and its deduced amino acidsequence (SEQ ID NO: 6);

FIGS. 1D-1 to 1D-9 show the nucleotide sequence of the D15 gene from H.influenzae non-typeable SB33 (SEQ ID NO. 7) and its deduced amino acidsequence (SEQ ID NO: 8);

FIGS. 1E-1 to 1E-8 show the nucleotide sequence of the D15 gene from H.influenzae non-typeable PAK 12085 (SEQ ID NO. 9) and its deduced aminoacid sequence (SEQ ID NO: 10);

FIGS. 1F-1 to 1F-29 show an alignment of the nucleotide sequences of theD15 genes (SEQ ID NOS: 1, 3, 5, 7 and 9) obtained from different H.influenzae isolates (typeable, Ca, Eagan and MinnA; nontypeable SB33 andPAK 12085) and the consensus sequence (SEQ ID NO.:55) for the D15 gene;

FIG. 2 shows restriction maps of clones pUC19/D15 (Ca), DS-712-2-1(Eagan), DS-691-1-5 (MinnA), JB-1042-5-1 (SB33), and JB-1042-9-4 (PAK12085). H=HindIII; R=EcoRI; S=Sau3A I; and Xb=XbaI;

FIGS. 3A and 3B show an alignment of the amino acid sequences of D15outer membrane proteins (SEQ ID NOS: 2, 4, 6, 8 and 10) obtained fromdifferent H. influenzae isolates (typeable, Ca, Eagan and MinnA;nontypeable, SB33 and PAK 12085). Amino acids are represented by theconventional one-letter code. The Ca D15 sequence is used as referenceand the dots indicate amino acid residues which are identical to thoseof the Ca D15 outer membrane protein;

FIG. 4 shows the construction of a plasmid (DS-880-1-2) expressingfull-length SB33 D15 (rD15) from the strong inducible T7 promoter;

FIG. 5A and 5B show an SDS-PAGE analysis of native D15 affinity-purifiedfrom H. influenzae strain 30;

FIG. 6 shows an SDS-PAGE analysis of sequential fractions obtainedduring the purification of the full-length rD15 expressed in E. colicontaining plasmid DS-880-1-2;

FIG. 7 shows guinea pig IgG antibody responses to full length rD15. Thearrows indicate the immunization schedule. Bleeds were taken at 0, 2, 4,6 and 8 weeks. The bars represent the standard deviation;

FIGS. 8A and 8B show mouse IgG antibody responses to full length rD15.The arrows indicate the immunization schedule. Bleeds were taken at 0,1, 4, 5 and 7 weeks. The bars represent the standard deviation;

FIG. 9 shows an SDS-PAGE analysis of the N-terminal rD15 fragmentpurified from GST-(D15 fragment) fusion protein. Lanes: 1, prestainedlow molecular weight markers (14 kDa, 21 kDa, 31 kDa, 45 kDa, 68 kDa, 97kDa); 2, GST standard; 3, GST-(D15 fragment) fusion protein; 4, fusionprotein cleaved by thrombin; 5, N-terminal rD15 fragment; 6, GST; 7, lowmolecular weight markers;

FIGS. 10A and 10B show guinea pig IgG antibody response to N-terminalrD15 fragment. The arrows indicate the immunization schedule. Bleedswere taken at 2, 4, 6 and 8 weeks. The bars represent the standarddeviation; and

FIG. 11 shows the hydrophilicity plot of D15 established by using awindow average across 7 residues according to Hope, 1986.

GENERAL DESCRIPTION OF THE INVENTION

Any Haemophilus strains that have D15 genes may be conveniently used toprovide the purified and isolated nucleic acid molecules (which may bein the form of DNA molecules), comprising at least a portion coding fora D15 outer membrane protein as typified by embodiments of the presentinvention. Such strains are generally available from clinical sourcesand from bacterial culture collections, such as the American TypeCulture Collection. H. influenzae strains may include types a, b and cstrains, non-typeable strains and other bacteria that produce a D15protein, fragment or analog thereof. Appropriate strains of Haemophilusinclude:

H. influenzae type b strain Ca;

H. influenzae type b strain MinnA;

H. influenzae type b strain Egan;

H. influenzae non-typeable b strain SB33; or

H. influenzae non-typeable b strain PAK 12085.

In this application, the term D15 outer membrane protein is used todefine a family of D15 proteins which includes those having naturallyoccurring variations in their amino acid sequences as found in variousstrains of, for example, Haemophilus. The purified and isolated DNAmolecules comprising at least a portion coding for D15 outer membraneprotein of the present invention also include those having naturallyoccuring variations in their nucleic acid sequences as found in variousstrains of, for example Haemophilus and those DNA molecules encodingfunctional analogs of D15 outer membrane protein. In this application, afirst protein is a functional analog of a second protein if the firstprotein is immunologically related with and/or has the same function asthe second protein. The functional analog may be, for example, afragment of the protein or a substitution, addition or deletion mutantthereof.

In aspects of the present invention, the D15 gene was isolated from H.influenzae type b strain Ca as shown in FIG. 1A; H. influenzae type BEagan, FIG. 1B; H. influenzae type b MinnA, FIG. 1C; non-typeable H.influenzace SB33, FIG. 1D; non-typeable H. influenzae PAK 12085, FIG.1E. A comparison of the nucleic acid sequences of the D15 genes and ofthe deduced amino acid sequences of the D15 outer membrane proteins fromthese strains of H. influenzae showed the genes and proteins to behighly conserved (FIGS. 1F and 3). The consensus sequence (SEQ ID NO:55) for the D15 gene is shown in FIG. 1F.

The purified and isolated DNA molecules comprising at least a portioncoding for a D15 outer membrane protein of a species of Haemophilus,typified by the embodiments described herein, are advantageous as:

nucleic acid probes for the specific identification of Haemophilusstrains in vitro or in vivo

the products encoded by the DNA molecules are useful as diagnosticreagents, antigens for the production of Haemophilus-specific antisera,for vaccination against the diseases caused by species of Haemophilusand detecting infection by Haemophilus; and

peptides corresponding to portions of the D15 outer membrane protein astypified by the embodiments described herein are advantageous asdiagnostic reagents, antigens for the production of Haemophilus-specificantisera, for vaccination against the diseases caused by species ofHaemophilus and for detecting infection by Haemophilus.

Reference will now be made in detail to the presently preferredembodiments of the invention, which together with the followingExamples, serve to explain the principle of the invention. For clarityof disclosure, and not by way of limitation, the detailed description ofthe invention is divided into the following sections:

(i) The DNA Sequences Coding for the Outer Membrane Protein D15 from H.influenzae Type b Ca Strain.

A clone producing the outer membrane protein designated D15 of H.influenzae type b (Hib) was isolated by screening a genomic library withH. influenzae type b OMP-specific polyclonal antibodies as previouslydescribed by Berns and Thomas 1965; Thomas and Rossi 1986. The DNAfragment encoding the D15 protein was isolated, subcloned into pUC19 toproduce pUC19/D15 (FIG. 2) and used to transform E. coli HB101 asdescribed in Example 1. Plasmid DNA was prepared from two individualcolonies of E. coli HB101 containing the pUC19/D15 plasmid. Sequencingwas performed on an ABI DNA sequencer model 370A using dye-terminatorchemistry and oligonucleotide primers which had been synthesized on anABI DNA synthesizer model 380B, and purified by chromatography.Nucleotide sequence analysis of the D15 gene revealed that it contains aputative promoter and an open reading frame encoding 789 amino acids(FIG. 1A).

The first 19 amino acid residues of the translated open reading frameform a typical leader sequence as found in other H. influenzae type bouter membrane proteins, such as P1 and P2. The N-terminal sequence ofimmuno-affinity purified native D15 antigen was determined by automatedEdman degradation using the ABI 477A protein sequencer and was found tobe Ala-Pro-Phe, which is identical to the N-terminal amino acid sequenceAla-Pro-Phe-Val-Ala-Lys-(SEQ ID NO: 11) predicted from an analysis ofthe sequence of the D15 gene presented in FIG. 1A.

(ii) The Sequence pf D15 Genes from Other H. influenzae Strains

D15 genes were isolated from other H. influenzae strains by screeningthe chromosomal libraries of H. influenzae type b strains Eagan, Minn Aand the non-typeable H. influenzae (NTHi) strains SB33 and PAK 12085, asdescribed in Examples 2, 3 and 4. Hybridization-positive clones wereplated and submitted to a second round of screening. The restrictionmaps of the clones obtained are shown in FIG. 2. The nucleotidesequences of the D15 genes were determined for all these clones (FIGS.1B to 1E) and their derived amino acid sequences compared (FIG. 3). TheD15 amino acid sequences of the three H. influenzae type b strains wereidentical and only a few amino acid differences were observed in theamino acid sequence of the D15 protein from the non-typeable strains(FIG. 3).

(iii) Expression of D15 and its Fragments in E. coli.

Since D15 is expressed in small quantities by strains of H. influenzae,it is advantageous to either express this antigen as a recombinantprotein in a heterologous system, such as E. coli, or to modify the H.influenzae organism to enhance native D15 expression. The Hind III/EcoRI fragment of H. influenzae type b Ca strain DNA encoding the fulllength D15 protein was expressed in pUC19 but not pUC18, suggesting thatthe lac promoter is helping to express the D15 gene in E. coli, eventhough the native D15 gene promoter is present. The T7 expression systemis a tightly controlled, inducible system which has great utility inexpression of heterologous proteins in E. coli The T7 expression systemis described in U.S. Pat. No. 4,952,496. Clones were, therefore,constructed which utilize the T7 system to express a mature D15 proteinthat contains an additional methionine residue at the amino terminus.The D15 signal sequence was removed during this construction process. Afull length recombinant D15 (termed rD15) was expressed in inclusionbodies which allow the D15 protein to be readily purified. The D15 genesfrom H. influenzae type b strain Ca and H. influenzae non-typeable SB33strain have been expressed at high levels in E. coli using the T7 systemto permit production of large quantities of rD15 protein. Theconstruction of clone DS-880-1-2 which expresses the SB33 D15 gene isdescribed herein (see FIG. 4 and Example 5). The rD15 protein wasimmunologically similar to its native counterpart isolated from H.influenzae typeable and non-typeable strains (see below). Thus, rD15 maybe used as a cross-reactive antigen in a diagnostic kit to detect many,if not all, strains of H. influenzae and other bacteria that produce aD15 outer membrane protein or analog thereof. Alternatively, rD15 can beused as an antigen to specifically detect the presence of H. influenzaein a sample.

A truncated D15 fragment was expressed in E. coli as a fusion proteinwith glutathione S-transferase (GST), as described in Example 6. Theconstruction was designed to express the N-terminal fragment of the D15protein. The fusion protein was expressed at high levels from a pGEX-2Tconstruction and the N-terminal fragment was cleaved from the GSTcarrier protein by treatment with thrombin. This procedure generated amolecule termed the N-terminal rD15 fragment which encompasses aminoacids 63-223 of the D15 protein. This N-terminal rD15 fragment washighly immunogenic and elicited protective antibodies against challengewith live H. influenzae.

(iv) Purification of Native D15 from H. influenzae Cell Paste.

The present invention also provides a method to prepare purified nativeD15 protein from H. influenzae. The protein is extracted andaffinity-purified from the cell pastes of either H. influenzae typeableor non-typeable isolates by a procedure involving the dissolution of theprotein in an aqueous detergent solution (see Example 13). The nativeD15 protein from a non-typeable H. influenzae strain 30 was solubilizedwith a 50 mM Tris-HCl/0.5% Triton X-100/10 mM EDTA buffer, pH 8.0 andfurther purified on a D15-specific monoclonal antibody affinity column(FIG. 5A). An 80 kDa protein was eluted from the column with 50 mMdiethylamine, pH 12.0 and shown to react with a D15-specific monoclonalantibody on immunoblot analysis (FIG. 5B). The native D15 is also highlyimmunogenic in experimental animals. Rabbit anti-D15 antisera reactedwith all H. influenzae isolates as determined by immunoblot analyses.

(v) Purification of a Full-length Recombinant D15 Protein Expressed inE. coli.

A full-length recombinant D15 (rD15) protein was expressed in inclusionbodies in E. coli. As shown in FIG. 6, purification of rD15 inclusionbodies was achieved by a sequential extraction of the E. coli celllysate with 50 mM Tris-HCl1, pH 8.0, then 50 mM Tris containing 0.5%Triton X-100 and 10 mM EDTA, pH 8.0. After centrifugation, more than 95%of the proteins in the resulting pellet was an 80 kDa protein bySDS-PAGE analysis, that reacted with a D15-specific monoclonal antibodyon an immunoblot. The N-terminal sequence of the rD15 was found to beMet-Ala-Pro-Phe-Val-Lys-Asp-(SEQ ID NO: 54) which is identical to thepredicted amino acid sequence.

The rD15 inclusion bodies were solubilized with a mixture of PBS, 0.5%Triton X-100, 10 mM EDTA and 8 M urea (see Example 8). After dialysisagainst PBS to remove urea, more than 80% of the D15 protein remainedsoluble. This soluble rD15 antigen was used for the immunogenicitystudies described below. From shake-flask experiments, it was estimatedthat about 10 mg of soluble rD15 protein was obtained from 1 L of E.coli bacterial culture. It is clear that growing the recombinant E. colistrains under optimised fermentation conditions significantly increasethe level of rD15 production.

(vi) Immunogenicity of the Full-length Recombinant D15 Protein (rD15)

The immunogenicity of the full-length rD15 protein was studied in guineapigs and mice. Using the immunization protocols described in FIG. 7, a15 μg dose of rD15 induced high IgG titers in guinea pigs whenadministered in the presence of either Preund's adjuvant or AlPO₄. Inthe mouse dose-response study, the protein appeared to be immunogenic ata dose as low as 5 μg in either Freund's adjuvant (FIG. 8A) or AlPO₄(FIG. 8B).

The protective ability of rD15 against H. influenzae type b infectionwas examined in the infant rat model of bacteremia essentially asdescribed by Loeb (1987). Thus, infant rats passively immunized withguinea pig anti-rD15 antisera were significantly less bacteremic thancontrols injected with pre-bleed sera, which is consistent with theprevious report by Thomas et al. (1990).

(vii) Purification and Characterization of the N-terminal rD15 Fragment.

The truncated rD15 fragment corresponding to the N-terminus of the D15protein (residues 22 to 223) as described in Example 6, was expressed inE. coli as a soluble protein fused to GST. The fusion protein (46 kDa)was readily extracted using phosphate buffered saline (PBS).Purification of the GST-D15 fragment fusion protein was achieved by asingle-step affinity purification process on a glutathione-Sepharose 4Bcolumn (FIG. 9, Lane 3). Cleavage of the 46 kDa fusion protein withthrombin yielded two fragments (FIG. 9, Lane 4), a 26 kDa protein whichcorresponded to a purified GST standard (FIG. 9, Lane 2), and a 20 kDapolypeptide which had the size expected for the N-terminal rD15 fragment(amino acid residues 63 to 223), respectively. Separation of these twoproteins was achieved by a second round of glutathione-Sepharose 4Baffinity chromatography. From shake-flask experiments, it was estimatedthat about 1 mg of purified N-terminal rD15 fragment was recovered from1 L of E. coli bacterial culture. It is clear that growing therecombinant E. coli strains under optimised fermentation conditions willsignificantly increase the level of N-terminal rD15 fragment production.

The identity of the 20 kDa polypeptide and the 26 kDa protein wasconfirmed by both immunoblotting and protein sequencing. The N-terminalsequence of the 20 kDa polypeptide was found to beNH₂-Ser-Leu-Phe-Val-Ser-Gly-Arg-Phe-Asp-Asp-Val-Lys-Ala-His-Gln-Glu-Gly-Asp-Val-Leu-Val-Val-Ser-(SEQID NO: 12), which corresponds to residues 63 to 85 of the primarysequence of D15. This result indicates that there is a spurious thrombincleavage site within the D15 sequence and that the first 42 amino acidsof the rD15 fragment are cleaved off during thrombin digestion. Thus,the final N-terminal rD15 fragment was 161 amino acids in lengthcorresponding to residues 63 to 223 of the primary sequence of D15. TheN-terminal sequence obtained for the 26 kDa protein(NH2-Met-Ser-Pro-Ile-Leu-Gly-Tyr-Trp-Lys-—SEQ ID NO: 13) confirmed thatit was GST.

(viii) Imogenicity of the N-terminal rD15 Fragment.

The immunogenicity of the N-terminal rD15 fragment was tested in guineapigs using various adjuvants. Using the immunization protocols describedin FIG. 10, a 10 μg dose of N-terminal rD15 fragment induced a goodbooster response in guinea pigs with almost all the adjuvants tested.The highest anti-D15 IgG titer was observed in the group of guinea pigsimmunized with N-terminal rD15 fragment in Freund's adjuvant. The secondbest adjuvant was Titermax (CytRx Inc.). The other two adjuvants, TPAD4(tripalmityl-Cys-Ser-Glu₄) and AlPO₄ were equally potent.

(ix) Protective Ability of the N-terminal rD15 Fragment Against H.influenzae Type b Challenge.

An in vivo challenge model for a assessing the protective abilities ofantigen against diseases caused by Haemophilus is the infant rat modelof bacteremia as described by Loeb 1987. The protective ability of theN-terminal rD15 fragment against H. influenzae type b challenge wasexamined in this rat model. As illustrated in Table 1, infant ratspassively immunized with rabbit anti-N-terminal rD15 fragment antiserashowed significantly lower bacteremia compared to those injected withpre-bleed sera.

Since passively transferred antisera against the N-terminal rD15fragment were found to be protective in the infant rat model ofbacteremia, it was of interest to identify the protective epitope(s) ofthis N-terminal rD15 fragment. The first nine overlapping peptides ofthe D15 protein as listed in Table 2 were chemically synthesized basedupon the amino acid sequence derived from the sequence of the D15 genefrom H. influenzae type b Ca (FIG. 1). These synthetic peptides wereassessed for their reactivities with either rabbit or guinea pigantisera raised against purified N-terminal rD15 fragment by ELISAs. Asshown in Table 3, both guinea pig and rabbit antisera reacted with acluster of D15 peptides, including peptides D15-P4 to D15-P8encompassing residues 93 to 209 of the D15 primary sequence.

Further studies were performed to determine whether the protectionagainst H. influenzae type b observed using rabbit anti-D15 antisera ininfant rats could be neutralized by D15 peptides. In the firstexperiment, a rabbit anti-N-terminal rD15 fragment antiserum wasinjected into a group of seven infant rats in the presence or absence ofa mixture of the nine D15 peptides (D15-P2 to D15-P10). Animals in thepositive control group were injected with the rabbit anti-N-terminalrD15 fragment antiserum mixed with purified D15 fragment and thenegative control group was injected with a mixture of the nine peptidesonly. As illustrated in Table 4, infant rats passively immunized with arabbit anti-N-terminal rD15 fragment antiserum (group #1) showed asignificantly lower bacteremia level (3%, p−1.2×10⁻⁷) compared to thosein the negative control group (group #4, 100%), which was consistentwith the previously obtained results. The protection mediated by therabbit anti-N-terminal rD15 fragment antiserum was largely neutralizedby the addition of purified N-terminal rD15 fragment (group #3, 64%), asindicated by the lack of significant difference in the bacteremia levelbetween group #3 and group #4 (p=0.09). Although the addition of themixture of nine D15 peptides only slightly neutralized the protectionconferred by the antiserum (group #2, 13%) as compared to group #1 (3%),the difference in bacteria counts between these two groups wasstatistically significant (p=0.0037).

To more clearly define the protective epitope(s) of the N-terminal rD15fragment, the above experiment was repeated with a mixture of fivepeptides (peptides D15-P4 to D15-P8) which were chosen for their strongreactivities with the rabbit anti-N-terminal rD15 fragment antiserum.The results obtained from this second experiment showed that theprotection observed using rabbit anti-N-terminal rD15 fragment (Table 5,group #1) was completely blocked by the addition of this mixture of fivepeptides (Table 5, group #2, 106%, p=0. 53×10⁻⁸) These results stronglyindicate that a cocktail of D15 synthetic peptides may be used asimmunogens to induce protective antibodies against H. influenzae.

(x) Epitope Prediction and Peptide Snythesis.

To map the immunodominant T-cell or B-cell epitopes of D15, overlappingsynthetic peptides covering the entire D15 protein sequence (Table 2—SEQ ID NO: 14 to 49) were synthesized using the t-Boc solid-phaaepeptide synthesis as described in Example 15. The peptides were chosenbased on their high index of hydrophillic β-turns estimated by secondarystructure prediction analysis (FIG. 11). Such peptides are likely to besurface-exposed and antigenic. Peptides more than 25 residues in lengthwere selected to better mimic native epitopes.

(xi) Identification and Characterization of Immunodominant Epitopes ofD15 using Synthetic Peptide.

To map the linear B-cell epitopes of D15, overlapping synthetic peptidesrepresenting the entire sequence of D15 were individually coated ontoELISA plates and probed with several anti-rD15 antisera as described inExample 19. The results are summarized in Table 6. Mouse antisera raisedagainst rD15 reacted with all D15 peptides, but the major epitopes werelocated within peptides D15-P8 (residues 180-209—SEQ ID NO: 21), D15-P10(residues 219-249—SEQ ID NO: 23), D15-P11 (residues 241-270—SEQ ID NO:24), and D15-P26 (residues 554-582—SEQ ID NO: 39), respectively. Rabbitanti-D15 antisera recognized only peptides D15-P4 (residues 93-122—SEQID NO: 17), D15-P14 (residues 304-333—SEQ ID NO: 27) and D15-P36(residues 769-798—SEQ ID NO: 49). Guinea pig antisera raised againstrD15 reacted with peptides D15-P2 (residues 45-72—SEQ ID NO: 15), D15-P4(residues 93-122—SEQ ID NO: 17), D1S-P6 (residues 135-164—SEQ ID NO:19), D15-P8 (residues 180-209—SEQ ID NO: 21), D15-P14 (residues304-333—SEQ ID NO: 27), D15-P27 (residues 577-602—SEQ ID NO: 40). Theimmunodominant linear B-cell epitopes of D15 were thus found to belocated within peptides D15-P4 (residues 93-122—SEQ ID NO: 17) andD15-P14 (residues 304-333—SEQ ID NO: 27), since these are the only twopeptides recognized by rD15-specific antisera from all three animalspecies. These results indicate that the peptides containing the linearB-cell epitope sequences described above can be used as target antigensin, for example, diagnostic kits to detect the presence of anti-D15 andanti-H. influenzae antibodies in samples.

(xii) Identification and Characterization of Immunodominant T-cellEpitopes of D15 using Synthetic Peptides.

The importance of cytokine networks in the immune and inflammatoryresponses in immunity and inflammation and their alteration in pathologyis becoming more evident as new members of the cytokine family areidentified and characterized. Mills et al. (1993) have recently reportedthat there is a rapid clearance of B. pertussis from the lungs of miceon challenge six weeks after respiratory infection or following twoimmunizations with the whole-cell pertussis vaccine. Spleen cells fromthese immunized mice were found to secrete high levels of IL-2 and IFN-γand low levels of IL-5 in the presence of pertussis antigen (pertussistoxoid, filamentous haemagglutinin (FHA) and pertactin). This resultsuggests that Th1 cell (T-cells producing high levels of IL-2 and IFN-γ)proliferation is very important for recovering from respiratoryinfection. The generation of Th1 and Th2 cell subsets is regulated bythe balance between different groups of cytokines, predominantly IL-12and IL-4 (Trinchieri, 1993). IL-12 and IL-4 are responsible for Th1 andTh2 cells differentiation, respectively. One of the roles of Th2 cellsin the immune system is to provide helper activity for eliciting highlevels of antigen-specific antibodies following immunization. Antigenscontaining Th1epitope(s) stimulate antigen-specific T-cells to producehigh levels of IL-2 and IFN-γ, whereas Th2 epitope(s) induce high levelsof IL-4 expression. Th0 epitope(s) stimulate the synthesis of IFN-γ andIL-4.

Little is known about the cellular immune response to outer membraneproteins of H. influenzae and its role in the protection against H.influenzae infection and diseases. To this end, the inventors performedstudies of the cellular response elicited in mice following rD15immunization. D15-specific T-cell epitopes were determined using D15peptides and T-cell lines obtained from five BALB/c mice immunized withrD15 (see Example 23). The lymphocyte proliferative responses of theD15-specific T-cell lines to overlapping D15 peptides were determined inconventional cytokine assays as described in Example 24. The resultssummarized in Table 7, revealed that stimulation only with certainsynthetic peptides elicited proliferative responses and the release ofspecific cytokines. Synthetic peptides corresponding to residues 114-143(D15-P5—SEQ ID NO: 18), 282-312 (D15-P13—SEQ ID NO: 26) and 577-602(D15-P27—SEQ ID NO: 40), and 219-249 (D15-P10—SEQ ID NO: 23), 262-291(D15-P12—SEQ ID NO: 25), 390-416 (D15-P18—SEQ ID NO: 31), 410-435(D15-P19—SEQ ID NO: 32) 554-582 (D15-P26—SEQ ID NO: 39), 596-625(D15-P28—SEQ ID NO: 41), 725-750 (D15-P34—SEQ ID NO: 47) and 745-771(D15-P35—SEQ ID NO: 48) were shown to be highly stimulatory forrD15-specific BALB/c Th0 cells and Th1 cells, respectively. Therefore,these immunodominant T-cell epitopes can be used as autologous carriersfor PRP, and/or OMP B-cell epitopes to enhance their immunogenicity. TheTh1 cell epitopes identified above may be useful in the H. influenzaevaccine formulations to induce H. influenzae-specific cellular immuneresponses.

(xiii) Immunogenicity of D15 Peptides.

To determine whether synthetic D15 peptides were immunogenic freepeptides were assessed individually for their immunogenicity. Rabbit andguinea pig anti-peptide antisera were tested for their reactivities withthe immunizing peptides as well as with native D15 and rD15 by ELISA andimmunoblotting. As shown in Table 8, all guinea pig anti-D15 peptideantisera except those raised against D15-P26 (SEQ ID NO: 39), D15-P29(SEQ ID NO: 42), D15-P30 (SEQ ID NO: 43) and D15-P31 (SEQ ID NO: 44)were shown to be immunogenic by ELISAs. The induction of high titers ofpeptide-specific IgG antibodies by free peptides clearly indicates thatmost peptides contain both a functional T-helper determinant and aB-cell epitope(s). In addition, these anti-peptide antisera recognizedD15 in the immunoblot assay. Since most peptides contain potentfunctional T-helper determinant(s) and induce strong IgG antibodyresponses in mammals, they are candidate immunogens for inclusion in anH. influenzae vaccine preparation. D15 peptide-specific antiseracross-reacted with D15 from non-typeable strains of H. influenzae asjudged by immunoblotting. This finding indicates that immunogenic D15peptides contain epitopes which are highly conserved among typeable andnon-typeable strains of H. influenzae. In addition, polyclonalantibodies against these epitopes are useful to detect H. influenzae inbiological samples.

Therefore, these conserved epitopes of D15 can be used eitherindividually or in combination to prepare cross-reactive syntheticimmunogens against typeable and non-typeable strains of H. influenzaeand other bacteria that produce D15 protein, a fragment or an analogthereof. Peptides described above can be further polymerized, ormodified with lipids as lipopeptides, or linked to polysaccharidesincluding PRP as synthetic glycopeptide or lipoglycopeptide conjugatesto produce alternate vaccines. These vaccines can be used to immunizeagainst diseases caused by H. influenzae when administered to mammals,for example, by the intramuscular or parenteral route, or when deliveredusing microparticles, capsules, liposomes and targeting molecules, suchas toxins or fragments thereof, and antibodies, to cells of the immunesystem or mucosal surfaces.

(xiv) Utility of D15 as Carrier Protein for the Production ofGlycoconjugates.

To determine whether D15 may serve both as a protective antigen and acarrier, D15-PRP conjugation experiments were performed as described inExample 14. The D15-PRP conjugates were found to be highly immunogenicin rabbits and able to elicit both anti-D15 and anti-PRP IgG antibodyresponses as judged by D15-specific ELISA and PRP-BSA immunoassay (Table9). These results clearly demonstrate the practical utility of D15 as acarrier protein for glycoconjugation technology.

In preferred embodiments of the present invention, the carrier functionof D15 can be generally utilized to prepare chimeric molecules andconjugate vaccines against pathogenic bacteria, including encapsulatedbacteria. Thus, the glycoconjugates of the present inventions may beapplied to vaccinations to confer protection against infection with anybacteria having polysaccharide antigens, including, for example,Haemophilus influenzae, Streptococcus pneumoniae, Escherichia coli,Neisseria meningitidis, Salmonella typhi, Streptococcus mutans,Cryptococcus neoformans, Klebsiella, Staphylococcus aureus andPseudomonas aeruginosa.

In another embodiment, the carrier function of D15 may be used, forexample, to induce immunity toward abnormal polysaccharides of tumorcells, or to produce anti-tumor antibodies that can be conjugated tochemotherapeutic or bioactive agents.

Accordingly, the present invention provides the primary sequence and thepreparation of an antigen (D15) of H. influenzae that can be used in theprevention and diagnosis of diseases caused by Haemophilus. Inparticular, the inventors discovered that recombinant D15 or itsfragments, can elicit protective antibody responses against live H.influenzae type b bacteria challenge. Thus, the present inventions haveutility in vaccines. The invention also discloses the nucleotidesequences of the D15 genes isolated from both H. influenzae type bstrains and non-typeable isolates. The DNA segments encoding D15 aredisclosed and show minor polymorphism in both their nucleotide andderived amino acid sequences (FIGS. 1F and 3). These DNA segments may beused to provide an immunogen essentially free from other H. influenzaeantigens (such as PRP and lipooligosaccharides (LOS)) through theapplication of recombinant DNA technology. The present disclosurefurther provides novel techniques which can be employed for preparingessentially pure D15 or fragments thereof, as well as functionalanalogs. The recombinant D15 protein, fragment or analog thereof, may beproduced in a suitable expression system, such as E. coli, HaemophilusBordetella, Bacillus, Fungi, Yeast, Baculovirus, Poxvirus, vaccinia ormammalian expression systems.

In one embodiment, the present invention concerns the process ofpreparing vaccine compositions which include purified recombinant D15protein (rD15) or rD15 fragments that are immunologically cross-reactivewith native D15. In particular, the gene coding the entire D15 proteinand a DNA segment encoding an N-terminal rD15 fragment fused to theglutathione-S-transf erase gene have been constructed and expressed inE. coli. The expressed rD15 protein and its fragments were found tocross-react immunologically with the native D15 antigen isolated fromboth typeable and non-typeable H. influenzae isolates and thus representcross-reactive immunogens for inclusion in a vaccine against diseasescaused by H. influenzae. Furthermore, Haemophilus convalescent serumrecognized D15 purified from H. influenzae as described herein, rD15 andN-terminal rD15 fragment.

In another embodiment, the present invention provides a gene coding forthe outer membrane protein D15 from H. influenzae having the specificnucleotide sequences described herein or ones substantially homologousthereto (i.e. those which hybridize under stringent conditions to suchsequences), for genetically engineering hybrids or chimeric proteinscontaining a D15 fragment fused to another polypeptide or protein or apolysaccharide, such as H. influenzae outer membrane proteins, forexample, P1, P2, or P6 or PRP. As a result, the hybrids, chimericproteins or glycoconjugates may have higher protectivity against H.influenzae than D15, or P1, or P2, or P6, or PRP alone.

Thus, D15 outer membrane protein can function both as a protectiveantigen and as a carrier in a conjugate vaccine to provide autologousT-cell priming, wherein the hapten part of the conjugate is the capsularpolysaccharide moiety (PRP) of H. influenza. This D15-carbohydrateconjugate can elicit antibodies against both PRP and D15, and thusshould enhance the level of protection against H. influenzae-relateddiseases, especially in infants.

In another embodiment, the present invention comprises an essentiallypure form of at least one protein or peptide containing an amino acidsequence corresponding to at least one antigenic determinant of D15,which peptide is capable of eliciting polyclonal antibodies against H.influenzae in mammals. These D15-specific antibodies are useful in testkits for detecting the presence of H. influenzae in biological samples.The peptides can have, for example, the amino acid sequencescorresponding to residues 20-49, 45-74, 68-99, 93-122, 114-143, 135-164,157-187, 180-209, 199-228, 219-249, 241-270, 262-291, 282-312, 304-333,325-354, 346-375, 367-396, 390-416, 410-435, 430-455, 450-477, 471-497,491-516, 511-538, 532-559, 554-582, 577-602, 596-625, 619-646, 641-666,662-688, 681-709, 705-731, 725-750, 745-771, 769-798 (SEQ ID NOS: 14 to49) of the D15 protein of the H. influenzae type b Ca strain,respectively, as set forth in Table 2 below, or any portion, variant ormutant thereof which retains immunogenicity.

In yet another embodiment, the present invention provides pure nativeD15 protein, extracted and chromatographically purified from cultures ofH. influenzae typeable or non-typeable isolates. The novel proceduresinvolves extraction of the D15 protein from cell paste by techniquesknown for other outer membrane proteins, with an aqueous detergentsolution, followed by purification by centrifugation and chromatography.The purified native D15 antigen can be used to immunize mammals againstdiseases caused by H. influenzae, for example, by the intramuscular orthe parenteral routes, or by delivering it using microparticles,capsules, liposomes and targeting molecules, such as toxins or fragmentsthereof, and antibodies.

Another aspect of the present invention is that the D15 outer membraneprotein, fragments or analogs thereof or peptides corresponding toportions of D15 may be components of a multivalent vaccine againstotitis media. This multivalent vaccine comprises at least oneimmunogenic determinant of D15 as described herein, along with at leastone protective antigen isolated from Strentococcus lneumoniae,Branhamella (Moroxella) catarrhalis, Stalphlococcus aureus, orrespiratory syncytial virus, in the presence or absence of adjuvant.

The D15 peptides (Table 2) or any portion, variant or mutant thereof,can easily be synthesized either manually or with a commerciallyavailable peptide synthesizer, such as the Applied Biosystems Model 430Asynthesizer.

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis, and treatment of diseases caused byHaemophilus infections, and the generation of immunological reagents. Afurther non-limiting discussion of such uses is further presented below.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable for use as vaccines, may be preparedfrom immunogenic D15 outer membrane protein, fragments or analogsthereof and/or peptides corresponding to portions of D15 as disclosedherein. The vaccine elicits an immune response which producesantibodies, including anti-D15 outer membrane protein antibodies andantibodies against D15 that are opsonizing or bactericidal. Should thevaccinated subject be challenged by Haemophilus, the antibodies bind tothe D15 outer membrane protein and thereby inactivate the bacterium.Opsonizing and bactericidal antibodies represent examples of antibodiesuseful in protection against disease.

Vaccines containing peptides are generally well known in the art, asexemplified by U.S. Pat. Nos. 4,601,903; 4,599,231; 4,599,230; and4,596,792; all of which references are incorporated herein by reference.As to any further reference to patents and references in thisdescription, they are as well hereby incorporated by reference withoutany further notice to that effect. Vaccines may be prepared asinjectables, as liquid solutions or emulsions. The D15 outer membraneprotein, fragments or analogs thereof or peptides corresponding toportions of D15 may be mixed with physiologically-acceptable excipientswhich are compatible with the D15 outer membrane protein, fragments,analogs or peptides. Excipients may include, water, saline, dextrose,glycerol, ethanol, and combinations thereof. The vaccine may furthercontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents, or adjuvants to enhance theeffectiveness of the vaccines. Methods of achieving adjuvant effect forthe vaccine includes use of agents, such as aluminum hydroxide orphosphate (alum), commonly used as 0.05 to 0.1 percent solution inphosphate buffered saline. Vaccines may be administered parenterally, byinjection subcutaneously or intramuscularly. Alternatively, other modesof administration including suppositories and oral formulations may bedesirable. For suppositories, binders and carriers may include, forexample, polyalkalene glycols or triglycerides. Oral formulations mayinclude normally employed incipients such as, for example,pharmaceutical grades of saccharine, cellulose, magnesium carbonate andthe like. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders andcontain 10-95% of the D15 outer membrane protein, fragment analogsand/or peptides.

The vaccines are administered in a manner compatible with the dosageformulation, and in an amount which is therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theindividual's immune system to synthesize antibodies, and if needed, toproduce a cell-mediated immune response. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner. However, suitable dosage ranges are readily determinableby one skilled in the art and may be of the order of micrograms of theD15 outer membrane protein, analog, fragment and/or peptides. Suitableregimes for initial administration and booster doses are also variable,but may include an initial administration followed by subsequentadministrations. The dosage of the vaccine may also depend on the routeof administration and varies according to the size of the host.

The nucleic acid molecules encoding the D15 outer membrane protein ofthe present invention may also be used directly for immunization byadministration of the DNA directly, for example, by injection forgenetic immunization or by constructing a live vector, such asSalmonella, BCG, adenovirus, poxvirus or vaccinia. A discussion of somelive vectors that have been used to carry heterologous antigens to theimmune system are discussed in, for example, O'Hagan (1992). Processesfor the direct injection of DNA into test subjects for geneticimmunization are described in, for example, Ulman et al. (1993).

The use of peptides in vivo may first require their chemicalmodification since the peptides themselves may not have a sufficientlylong serum and/or tissue half-life. Such chemically modified peptidesare referred to herein as peptide analogs. The term peptide analogextends to any functional chemical equivalent of a peptide characterizedby its increased stability and/or efficacy in vivo or in vitro inrespect of the practice of the invention. The term peptide analog isalso used herein to extend to any amino acid derivative of the peptidesas described herein. Peptide analogs contemplated herein are produced byprocedures that include, but are not limited to, modifications to sidechains, incorporation of unnatural amino acids and/or their derivativesduring peptide synthesis and the use of cross-linkers and other methodswhich impose conformational constraint on the peptides or their analogs.

Examples of side chain modifications contemplated by the presentinvention include modification of amino groups, such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidation with methylacetimidate; acetylation with aceticanhydride; carbamylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzenesulfonic acid (TNBS); alkylation of amino groups with succinic anhydrideand tetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxa-5′-phosphate followed by reduction with NaBH₄.

The guanidino group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viao-acylisourea formation followed by subsequent derivatisation, forexample, to a corresponding amide.

Sulfhydryl groups may be modified by methods, such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of mixed disulphides with other thiol compounds;reaction with maleimide; maleic anhydride or other substitutedmaleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid,phenylmercury chloride, 2-chloromercuric-4-nitrophenol and othermercurials; carbamylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides. Tryosine residuesmay be altered by nitration with tetranitromethane to form a3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids.

2. Immunoassays

The D15 outer membrane protein, analog, fragment and/or peptides of thepresent invention are useful as antigens in immunoassays, includingenzyme-linked immunosorbent assays (ELISA), RIAs and other non-enzymelinked antibody binding assays or procedures known to the art for thedetection of anti-bacterial, Haemophius D15 and/or peptide antibodies.In ELISA assays, the D15 outer membrane protein, fragment or analogsthereof and/or peptides corresponding to portions of D15 outer membraneprotein are immobilized onto a selected surface, for example, a surfaceexhibiting a protein affinity, such as the wells of a polystyrenemicrotiter plate. After washing to remove incompletely adsorbed D15outer membrane protein, analog, fragment and/or peptides, a nonspecificprotein, such as bovine serum albumin (BSA) or casein, that is known tobe antigenically neutral with regard to the test sample may be bound tothe selected surface. This allows for blocking of nonspecific adsorptionsites on the immobilizing surface and thus decreases the backgroundcaused by nonspecific bindings of antisera onto the surface. Normally,the peptides employed herein are in the range of 12 residues and up andpreferably 14 to 30 residues.

The immobilizing surface is then contacted with a sample such asclinical or biological materials to be tested in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents, such as BSA, bovine gamma globulin (BGG)and/or phosphate buffered saline (PBS)/Tween. The sample is then allowedto incubate for from 2 to 4 hours, at temperatures, such as of the orderof 25° to 37° C. Following incubation, the sample-contacted surface iswashed to remove non-immunocomplexed material. The washing procedure mayinclude washing with a solution such as PBS/Tween, or a borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound D15 outer membrane protein, analog, fragment and/orpeptides, and subsequent washing, the occurrence, and even amount, ofimmunocomplex formation may be determined by subjecting theimmunocomplex to a second antibody having specificity for the firstantibody. If the test sample is of human origin, the second antibody isan antibody having specificity for human immunoglobulins and, ingeneral, IgG. To provide detecting means, the second antibody may havean associated activity, such as an enzymatic activity that willgenerate, for example, a color development upon incubating with anappropriate chromogenic substrate. Quantification may then achieved bymeasuring the degree of color generation using, for example, a visiblespectra spectrophotometer.

3. Use of Sequnces as Hybridization Probes

The nucleotide sequences of the present invention comprising thesequence of the D15 outer membrane protein, now allow for theidentification and cloning of the D15 outer membrane protein genes fromany species of Haemophilus and other bacteria that have genes encodingD15 outer membrane proteins.

The nucleotide sequences comprising the sequence encoding the D15 outermembrane protein of the present invention are useful for their abilityto selectively form duplex molecules with complementary stretches ofother D15 genes. Depending on the application, a variety ofhybridization conditions may be employed to achieve varying degrees ofselectivity of the probe toward the other D15 genes. For a high degreeof selectivity, stringent conditions are used to form the duplexes, suchas low salt and/or high temperature conditions, such as provided by 0.02M to 0. 15 M NaCl at temperatures of between about 50° C. to 70° C. Forsome applications, less stringent hybridization conditions are requiredsuch as 0.15 M to 0.9 M salt, at temperatures ranging from between about20° C. to 55° C. Hybridization conditions can also be rendered morestringent by the addition of increasing amounts of formamide, todestabilize the hybrid duplex. Thus, particular hybridization conditionscan be readily manipulated, and will generally be a method of choicedepending on the desired results.

In a clinical diagnostic embodiment, the nucleic acid sequences of theD15 outer membrane protein genes of the present invention may be used incombination with an appropriate means, such as a label, for determininghybridization. A wide variety of appropriate indicator means are knownin the art, including radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of providing a detectable signal. Insome diagnostic embodiments, an enzyme tag, such as urease, alkalinephosphatase or peroxidase, instead of a radioactive tag may be used. Inthe case of enzyme tags, calorimetric indicator substrates are knownwhich can be employed to provide means visible to the human eye orspectrophotometrically, to identify specific hybridization with samplescontaining D15 gene sequences.

The nucleic acid sequences of D15 genes of the present invention areuseful as hybridization probes in solution hybridizations and inembodiments employing solid-phase procedures. In embodiments involvingsolid phase procedures, the test DNA (or RNA) from samples, such asclinical samples, including exudates, body fluids (e. g., serum,amniotic fluid, middle ear effusion, sputum, bronchoalveolar lavagefluid) or even tissues, is adsorbed or otherwise affixed to a selectedmatrix or surface. The fixed, single-stranded nucleic acid is thensubjected to specific hybridization with selected probes comprising thenucleic acid sequences of the D15 genes or fragments thereof of thepresent invention under desired conditions. The selected conditions willdepend on the particular circumstances based on the particular criteriarequired depending on, for example, on the G+C contents, type of targetnucleic acid, source of nucleic acid, size of hybridization probe etc.Following washing of the hybridization surface so as to removenon-specifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label. The selected probeshould be at least 18 bp and may be in the range of 30 bp to 90 bp long.

4. Expression of the D15 Outer Membrane Protein Genes

Plasmid vectors containing replicon and control sequences which arederived from species compatible with the host cell may be used for theexpression of the D15 outer membrane protein genes in expressionsystems. The vector ordinarily carries a replication site, as well asmarking sequences which are capable of providing phenotypic selection intransformed cells. For example, E. coli may be transformed using pBR322which contains genes for ampicillin and tetracycline resistance and thusprovides easy means for identifying transformed cells. The pBR322plasmid, or other microbial plasmid or phage must also contain, or bemodified to contain, promoters which can be used by the microbialorganism for expression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used as atransforming vector in connection with these hosts. For example, thephage in lambda GEM™-11 may be utilized in making recombinant phagevectors which can be used to transform host cells, such as E. coliLE392.

Promoters commonly used in recombinant DNA construction include theβ-lactamase (penicillinase) and lactose promoter systems and othermicrobial promoters, such as the T7 promoter system. Details concerningthe nucleotide sequences of promoters are known, enabling a skilledworker to ligate them functionally with plasmid vectors. The particularpromoter used generally is a matter of choice depending upon the desiredresults. Hosts that are appropriate for expression of the transferrinreceptor genes, fragment analogs or variants thereof include E. coli,Bacillus, Haemophilus, Bordetella, fungi, yeast, or the baculovirus andpoxvirus expression systems may be used.

In accordance with an aspect of this invention, it is preferred to makethe D15 outer membrane protein, fragment or analog thereof byrecombinant methods, particularly since the naturally occurring D15protein as purified from culture of a species of Haemophilus may includeundesired contaminants, including trace amounts of toxic materials. Thisproblem can be avoided by using recombinantly produced D15 outermembrane protein in heterologous systems which can be isolated from thehost in a manner to minimize toxins in the purified material.Particularly desirable hosts for expression in this regard include Grampositive bacteria which do not have lipopolysaccharide (LPS) and are,therefore, endotoxin free. Such hosts include species of Bacillus andmay be particularly useful for the production of non-pyrogenic D15 outermembrane protein, fragments or analogs thereof.

BIOLOGICAL DEPOSITS

Certain plasmids that contain at least a portion coding for a D15 outermembrane protein from strains of Haemophilus influenzae that aredescribed and referred to herein have been deposited with the AmericanType Culture Collection (ATCC) located at 10801 University Boulevard,Manassas, Va. 20110-2209, USA pursuant to the Budapest Treaty and priorto the filing of this application. Samples of the deposited plasmidswill become available to the public upon grant of a patent based uponthis United States patent application. The invention described andclaimed herein is not to be limited in scope by plasmids deposited,since the deposited embodiment is intended only as an illustration ofthe invention. Any equivalent or similar plasmids that encode similar orequivalent antigens as described in this application are within thescope of the invention.

DEPOSITE SUMMARY ATCC Date Clone H. influenzae Designation DepositedDS-712-2-1 Eagan 75604 November 4, 1993 JB-1042-5-1 SB33 75606 November4, 1993 DS-880-1-2 Eagan 75605 November 4, 1993

The above disclosure generally describes the present inversion. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitations.Immunological and recombinant DNA methods may not be explicitlydescribed in this disclosure but are well within the scope of thoseskilled in the art.

EXAMPLES

Methods of molecular genetics, protein biochemistry, and immunology usedbut not explicitly described in this disclosure and these EXAMPLES areamply reported in the scientific literature and are well within theability of those skilled in the art.

Example 1

This Example illustrates the cloning and sequencing of the D15 genes.

Genomic DNA was purified from the Haemophilus influenzae type b strainCa by lysis of the bacteria with pronase and sodium dodecylsulphatefollowed by phenol extraction and isopropanol precipitation, accordingto Berns and Thomas, 1965. The DNA was then partially digested withEcoRI and the DNA fraction containing 6-10 kb fragments was isolatedfollowing electrophoresis in low-melting point agarose. These fragmentswere ligated into a lambda gtll Ampl vector (Thomas and Rossi, 1986) andcloned as a lysogen into E. coli strain BTA282. Recombinant clones wereselected for their ampicillin resistance conferred by the vector. Toidentify clones producing H. influenzae type b antigen, the clones werereplica-plated on nitrocellulose filters and duplicate colonies inducedfor expression by temperature switch to 42° C. for 2 hours. Colonieswere lysed by wetting the filters with 1% sodium dodecylsulphate (SDS).The filters were then placed into a chloroform-saturated atmosphere for15 min. The filters were then assayed by colony radioimmuno-assay usinga hyperimmune rabbit anti-H. influenzae type b antiserum absorbed withE. coli lysate for antigen expression. Clones shown by autoradiographyto be producing H. influenzae type b antigens were further purified andtheir replicates retested for reactivity with the hyperimmune anti-H.influenzae type b antiserum. The antiserum absorbed with 10¹⁰ intact H.influenzae type b bacteria (strain Ca) was used as negative control.

A number of clones were identified which reacted with the unabsorbed,but not with the absorbed antiserum and were further analysed. One ofthe clones, D15, was purified, grown and found to produce a H.influenzae type b antigen which migrated in sodium dodecyl sulphatepolyacrylamide gels with a M_(r) of about 80 kDa. Lysates from the D15clone were coupled to Sepharose™ 4B gel and used to affinity-purifyanti-D15 antibodies. This procedure is described by Thomas et al, 1990,except that the apparent M_(r) was initially reported to be about 103kDa. The affinity-purified antibodies to D15 were then shown to reactwith an M_(r) 80 kDa protein in an outer membrane protein preparation ofH. influenzae type b (sarcosyl insoluble fraction—Carlone et al, 1986).Radioimmuno dot blots and Western blots analyses of membranepreparations from both type b and nontypeable Haemophilus influenzaestrains showed that affinity-purified anti-D15 antibodies reacted withall isolates. These antibodies were found to be capable of passivelyprotecting infant rats from bacteraemia following intraperitonealinjection of live H. influenzae type b bacteria. The specificity of theprotection was confirmed by absorbing out the protective activity ofanti-D15 antibodies with a lysate of E. coli expressing D15 coupled toSepharose. The protection studies have been described in detail byThomas et al, 1990.

DNA from the lambda gtll Ampl D15 phage was isolated and a 5.7 kbfragment was released by EcoRI digestion. This fragment was subclonedinto pUC19 and the resulting plasmid transformed into E. coli HB101.Recombinant bacteria were found to produce the expected M_(r) 80 kDa H.influenzae type b antigen when examined by Western blotting. The insertDNA was then characterised by restriction endonuclease mapping. A 2.8 kbHindIII-EcoRI fragment was subcloned into pUC19 to generate plasmidpUC19/D15, which was transformed into E. coli HB101. The recombinantbacteria expressed a M_(r) 80 kD protein recognized by D15-specificantibodies. on Western blot analysis of E. coli lysates.

Plasmid DNA was prepared from two individual colonies of recombinant E.coli HB101 containing the pUC19/D15 plasmid using standard techniques.Oligonucleotide sequencing primers of 17-25 bases in length weresynthesized on the ABI model 380B DNA Synthesizer and purified bychromatography using OPC cartridges obtained from Applied BiosystemsInc., and used in accordance with the manufactures recommendations.Samples were sequenced using the ABI model 370A DNA Sequencer and dyeterminator chemistry according to manufacturers' protocols. Thissequence analysis indicated that the D15 gene contains an open readingframe encoding for 789 amino acids, including a putative signal sequence(FIG. 1). The derived amino acid sequence was found to contain thesequence of an internal peptide obtained by thrombin digestion of nativeD15 that had been chemically determined. The amino acid composition ofD15 derived from the D15 gene sequence was comparable (withinexperimental error) to that of the native protein as determined by aminoacid analysis.

Example 2

This Example illustrates the preparation of chromosomal DNA fromHaemophilus influenzae strains Eagan, MinnA, SB33, and PAK 12085.

H. influenzae strains were grown on Mueller-Hinton agar or in brainheart infusion broth as described by Harkness et al., 1992.

Eagan Chromosomal DNA

Bacteria from 50 mL of culture were pelleted by centrifugation at 5,000rpm, 20 minutes, 4° C. The pellet was resuspended in 25 mL TE (10 mMTris, 1 mM EDTA, pH 8.0) and 2×5 mL aliquots used for chromosomal DNApreparation. To each aliquot were added 0.6 mL of 10% sarkosyl and 0.15mL of 20 mg/mL proteinase K and the samples incubated at 37° C. for 1hour. The lysate was extracted once with Tris-saturated phenol (pH 8.0)and three times with chloroform:isoamyl alcohol (24:1). The aqueousphase was pooled for a final volume of 7 mL. Then, 0.7 mL of 3M sodiumacetate (pH 5.2) and 4.3 mL of isopropanol were added to precipitate theDNA which was spooled, rinsed with 70% ethanol, dried, and resuspendedin 1 mL of water.

MinnA. SB33, and PAK 12085 Chromosomal DNA

Bacteria from 50 mL of culture were pelleted by centrifugation at 5,000rpm for 15-20 minutes, at 4° C., in a Sorvall RC-3B centrifuge. The cellpellet was resuspended in 10 mL of TE (10 mM Tris-HCl, 1 mM EDTA, pH7.5), pronase was added to 500 μg/mL, and SDS to 1%. The sample wasincubated at 37° C. for about 4 hours until a clear lysate was obtained.The lysate was extracted once with Tris-saturated phenol, once withTris-saturated phenol/chloroform (1:1), and once with chloroform. Thefinal aqueous phase was dialysed for 24 hours against 2×500 mL of 1MNaCl at 4° C., changing the buffer once, and for 24 hours against 2×500mL of TE at 4° C., changing the buffer once. The final dialysate wasaliquotted for subsequent use.

Example 3

This Example illustrates the preparation of Haemophilus influenzaechromosomal libraries.

H. influenzae Eagan and PAK 12085 chromosomal DNAs were digested withSau3A I (0.5 unit/10 μg DNA) at 37° C. for 15 minutes andsize-fractionated by agarose gel electrophoresis. Gel slicescorresponding to DNA fragments of 15-23 kb were excised and DNA waselectroeluted overnight in dialysis tubing containing 3 mL of TAE (40 mMTris-acetate, 1 mM EDTA, pH 8.0) at 14 V. The DNA was precipitated twiceand resuspended in water before overnight ligation with EMBL3 BamH Iarms (Promega). The ligation mixture was packaged using the Lambda invitro packaging kit (Amersham) according to the manufacturer'sinstructions and plated onto E. coli NM539 cells. The library wastitrated, then amplified and stored at 4° C. under 0.3% chloroform.

MinnA chromosomal DNA (10 μg) was digested with Sau3A I (40 units) for2, 4, and 6 minutes then size-fractionated on a 10-30% sucrose gradientin TNE (20 mM Tris-HCl, 5 mM NaCl, 1 mM EDTA, pH 8.0). Fractionscontaining DNA fragments>5 kb were pooled and precipitated. In a secondexperiment, chromosomal DNA (2.6 μg) was digested with Sau3A I (4 units)for 1, 2, and 3 minutes and size-fractionated by preparative agarose gelelectrophoresis. Gel slices containing DNA fragments of 10-20 kb wereexcised and DNA extracted by a standard freeze/thaw technique. Thesize-fractionated DNA from the two experiments was pooled for ligationwith BamH I arms of EMBL3 (Promega). The ligation mix was packaged usingthe Gigapack II packaging kit (Amersham) and plated on E. coli LE392cells. The library was titrated, then amplified and stored at 4° C.under 0.3% chloroform.

SB33 chromosomal DNA (20 μg) was digested with Sau3A I (40 units) for 2,4, or 6 minutes and size-fractionated on a 10-30% sucrose gradient inTNE (20 mM Tris-HCl, 5 mM NaCl, 1 mM EDTA, pH 8.0). Fractions containingfragments>5 kb were pooled. In a second experiment, SB33 Chromosomal DNA(2 μg) was digested with Sau3A I (4 units) for 2, 4, or 6 minutes andsize-fractionated on a preparative agarose gel. Gel slices containingDNA fragments of 10-20 kb were excised and DNA extracted by a standardfreeze/thaw technique. The size-fractionated DNA from both experimentswas pooled for ligation with BamH I arms of EMBL3 (Promega). Theligation mix was packaged using the Gigapack II packaging kit and platedon LE392 cells. The library was titrated, then amplified and stored at4° C. under 0.3% chloroform.

Example 4

This Example illustrates the screening of the DNA libraries.

The Eagan, MinnA, SB33, and PAK 12085 DNA libraries were plated ontoLE392 cells on NZCYM plates using 0.7% top agarose in NZCYM as overlay.Plaque lifts onto nitrocellulose filters were performed followingstandard procedures, and filters were processed and hybridized with adigoxigenin-labelled D15 probe prepared according to the manufacturer'sspecifications (Boehringer Mannheim). The probe was the EcoR I/Hind IIIfragment from pUC19/D15 containing the entire Ca D15 gene (FIG. 2).Putative plaques were plated and submitted to a second round ofscreening using the same procedures. Phage DNA was prepared from 500 mLof culture using standard techniques, the insert DNA was excised by SalI digestion, and cloned into pUC to generate clones DS-712-2-1 (Eagan),DS-691-1-5 (MinnA), JB-1042-5-1 (SB33), and JB-1042-9-4 (PAK 12085),which are shown in FIG. 2.

The nucleotide sequences of the D15 genes from H. influenzae type bstrains Eagan and MinnA the non-typeable H. influenzae strains SB33 andPAK 12085 were determined and compared with that for strain Ca, as seenin FIGS. 1b, 1C, 1D, 1E and 1F. The derived amino acid sequence areshown in FIGS. 1B, 1C, 1D and 1E and are compared with the amino acidsequence of the D15 protein of H. influenzae type b Ca (FIG. 3).

Exanmle 5

This Example illustrates the expression of rD15 protein in E. coli.

A 2.8 kb fragment HindIII-EcoRI was subcloned into pUC19 and thispUC19/D15 plasmid was transformed into E. coli HB101. Upon induction,the positive clones expressed an 80 kDa protein which was recognized byD15-specific antisera on Western blot analysis. A HindIII-Pst I fragmentwas also subcloned into pUC19 and shown to express a 67 kDa protein.According to the restriction map, this 67 kDa protein corresponded to aC-terminal truncated D15 protein. on Western blot analysis, thistruncated D15 was still recognized by the D15-specific antisera.

Plasmids to express the D15 gene of the non-typeable strain SB33 in E.coli were constructed. Plasmid JB-1042-5-1 containing the SB33 D15 geneand its flanking regions, was digested with EcoRI and Hind III and the 3kb D15 insert subcloned into pUC to give plasmid pRY-60-1 (FIG. 4).Appropriate oligonucleotides were synthesized to restore the native D15sequence between the ATG codon of the expression plasmid pT7-7 and theBsrF I site within the D15 gene. These oligonucleotides had thefollowing sequence:

   Nde 5′-  TATGGCACCTTTTGTGGCAAAAGATATTCGTGTGGATGGTGTTCAAGGTGACCGTGGAAAACACCGTTTTCTATAAGCACACCTACCACAAGTTCCACTGAATCTACTTAGAATCAACAAACCGAGCAAGTTTACCTGTTCGTG - SEQ ID NO: 50TGGTTGTTTAGGCTCGTTCAAATGGACAAGCACGGCC-5′- SEQ ID NO: 51                               BsrF I

Plasmid pRY-60-1 was digested with EcoR I and BsrF I and the DNAfragment containing most of the D15 gene was purified. pUC was digestedwith EcoR I and Nde I and the vector fragment purified. Amulti-component ligation between the pUC and D15 fragments and theoligonucleotides generated plasmid DS-860-1-1 which contains a D15sequence without a promoter. pT7-7 was digested with Nde I and EcoR Iand the vector fragment purified. DS-860-1-1 was digested with Nde I andEcoR I and the D15 insert was purified and ligated with the T7-7 vectorgenerating plasmid DS-880-1-2 (FIG. 4).

The plasmid constructions were performed using E. coli JM109 as host.For expression, plasmid DS-880-1-2 was transformed into E. coliBL21/DE3, BL21/DE3/pLysS, or JM109/DE3 cells. Transformation of thecells was performed using either calcium chloride-treated competentcells or by electroporation using a BioRad electroporator. Transformedcells were grown in YT, M9, or NZCYM media and induced with IPTG orother inducing agents.

Example 6

This Example illustrates the construction and expression of the GST-D15fragment hybrid gene in E. coli.

A forward sense primer (primer 1) 5′-GGGGAATTCCAAAAGATGTTCGT (SEQ ID NO:52) and a reverse antisense primer CACGAATTCCCTGCAAATC-5′ (primer 7—SEQID NO: 53) were used to amplify a 2.8 Kb fragment HindIII-EcoRI of theD15 gene by the polymerase chain reaction that encodes the N-terminalamino acid residues 22 to 223 of the primary sequence of D15 protein(FIG. 1A). The nucleotide sequence of the 609bp amplified fragment wasconfirmed by DNA sequencing. The amplified gene segment was ligated intothe pGEX-2T vector downstream from the GST gene and transformed into E.coli TG-1. Colonies expressing the H. influenzae type b antigen werescreened with a rabbit anti-H. influenzae type b antiserum by colonyradioimmunoassay and isolated. The glutathione-S-transferase-D15fragment fusion protein produced by transformed E. coli was isolated byaffinity purification on glutathione agarose.

Example 7

This Example describes alternative expression systems for rD15.

The D15 gene or fragments thereof are also expressed in E. coli underthe control of other regulated promoters. The D15 gene or fragmentsthereof are expressed in the absence of the leader peptide, or in othercloning systems where toxicity of D15 expression to the host is notproblematic. The gene or fragments thereof are synthesized de novo or byemploying the polymerase chain reaction using suitable primers. Thesegenes are cloned into suitable cloning vectors or bacteriophage vectorsin E. coli or other suitable hosts directly when toxicity can beavoided. Expression systems are Gram-positive bacteria (such as Bacillusspecies), pox virus, adenovirus, baculovirus, yeast, fungi, BCG ormammalian expression systems.

Example 8

This Example illustrates the protocol for extraction and purification ofrD15 from E. coli expression system.

The cell pellet from a 250 mL culture, prepared as described in Example5, was resuspended in 40 mL of 50 mM Tris, pH 8.0, and disrupted bysonication (3×10 min, 70% duty circle). The extract was centrifuged at20,000×g and the resulting pellet saved. The initial pellet wasre-extracted with 40 mL of 50 mM Tris, 0.5% Triton X-100, 10 mM EDTA, pH8.0. The suspension was then sonicated for 10 minutes at 70% dutycircle. The extract was centrifuged at 300×g for 5 minutes. Theresulting supernatant was centrifuged again at 20,000×g for 30 min andthe resulting pellet was saved. The pellet was resuspended in 50 mMTris, 0.5% Triton X-100, 10 mM EDTA, pH 8.0. The suspension was thenmixed with PBS/8 M urea to a final urea concentration of 6 M. Thesolution was then dialyzed against PBS to remove urea. After dialysis,the solution was centrifuged at 300×g for 10 min., the supernatant wassaved and stored at 4° C.

Example 9

This Example demonstrates the purification of GST-(D15 fragment) fusionprotein using glutathione-Sepharose 4B affinity chromatography.

Five mg of GST-(D15 fragment) fusion protein crude extract, prepared asdescribed in Example 6, were dissolved in 5 mL of phosphate buffersaline (PBS) containing 1% Triton X-100. The solution was then loadedonto a Glutathione-Sepharose 4B column (2 mL) equilibrated with PBScontaining 1% Triton X-100. The run-through of the column was discarded.The column was washed with 20 mL of PBS and the GST-(D15 fragment)fusion protein was eluted with 50 mM Tris-HCl buffer, pH 8.0, containing5 mM glutathione. Elution was monitored by absorbance at 280 nm.Protein-containing fractions (2 mL/fraction) were collected and pooled.The purity of the protein was assessed by SDS-PAGE (FIG. 9, lane 3). Thefinal volume of the purified fusion protein was 6 mL.

Example 10

This Example illustrates the protocol used for thrombin digestion ofproteins to release the truncated D15 molecule.

The GST-(D15 fragment) fusion protein sample from Example 9 (0.1 to 0.5mg protein/mL) was dialyzed against 1 L of 50 mM Tris-HCl buffer (pH8.5) 3 times with at least 2 hour intervals at 4° C. to remove proteaseinhibitors. After dialysis, the solution was treated with human thrombin(Sigma) at a ratio of 1 mL of solution to 25 units of thrombin. Thecleavage reaction was carried out at 37° C. for 2 hr and analysed bySDS-PAGE (FIG. 9, lane 4). The reaction was stopped by placing thesolution in ice.

Example 11

This Example illustrates the procedure used for N-terminal rD15 fragmentpurification from GST using Glutathione-Sepharose 4B affinitychromatography.

A thrombin-digested GST-(D15 fragment) sample, prepared as described inExample 10, was loaded onto a Glutathione-Sepharose 4B column (2 mL)equilibrated with PBS containing 1% Triton X-100. The run-through of thecolumn containing the N-terminal rD15 fragment was saved. After washingthe column with 20 mL of PBS, the affinity column was regenerated byremoving GST using 50 mM Tris-HCl buffer, pH 8.0, containing 5 mMglutathione. The purity of rD15 fragment was analysed by SDS-PAGE (FIG.9, lane 5). This N-terminal rD15 fragment contains amino acids 63-223 ofthe D15 protein as a result of cleavage at the spacious thrombin siteshown in FIG. 1A.

Example 12

This Example illustrates the protocol used for the purification ofD15-specific polyclonal antibodies by affinity chromatography usingGST-(D15 fragment) fusion protein.

The recombinant GST-(D15 fragment) fusion protein, prepared as describedin Example 9, was conjugated to cyanogen bromide-activated Sepharose.The affinity column was then used to purify antibodies from a rabbithyperimmune anti-H. influenzae type b antiserum. The affinitypurified-antibodies were shown by immunoblotting to react with a 80 kDacomponent present in the lysates of E. coli transformed with pUC9/D15and in the lysates of several typeable and nontypeable H. influenzaeisolates. These results confirmed that the DNA segment encoding the D15fragment of the fusion protein was part of the open reading frame of theD15 gene.

Similarly, antisera raised against the recombinant fusion protein(Example 9) or the purified N-terminal rD15 fragment (Example 11)reacted with the D15 protein produced by H. influenzae strains (Example13).

Example 13

This Example describes the protocol used for the purification of nativeD15 from H. influenzae.

Cell paste of the non-typeable H. influenzae SB33 strain, prepared froma culture grown in brain heart infusion medium supplemented with NAD(2μg/mL) and HEMIN (2 μg/mL) at 37° C., as described in Panezutti, etal, 1993, was resuspended in 50 mM Tris-HCl, pH 8.0, containing 0.5%Triton X-100 and 10 mM EDTA (20 mL per 1 g of cell paste). The mixturewas stirred at room temperature for 2 hr, then centrifuged at 20,000×gfor 30 minutes. The D15 was located in the supernatant and furtherpurified.

Purification of native D15 was achieved by affinity chromatography usinga D15-specific monoclonal antibody (see Example 24). The D15 extract (25mL) was mixed with the affinity matrix (1 mL) at room temperature for 2hr. The mixture was packed into a column and the run-through fractionwas discarded. The column was washed sequentially with the followingbuffers: 50 mM Tris-HCl, pH 8.0, containing 0.5% Triton X-100 and 10 mMEDTA; 1 M HEPES buffer, pH 6.8; 50 mM Tris-HCl, pH 8.0, containing 0.5%Triton X-100 and 10 mM EDTA; and 10 mM phosphate buffer, pH 8.0. D15 wasthen eluted from the column with 3 mL of 50 mM diethylamine, pH 12.0 andthe protein solution was neutralized by 1 M HEPES, pH 6.8 ({fraction(1/10)} volume). The affinity-purified native D15 was analysed bySDS-PAGE and stored at −20° C.

Example 14

This Example describes the procedure used for the preparation of D15-PRPconjugates.

Haemophilus influenzae type b oligosaccharides (PRP) prepared bycontrolled acid hydrolysis were conjugated either with the purifiednative (Example 13) or recombinant D15 (Example 8) as well as with itsfragments (Example 11) using periodate oxidation as described in U.S.Pat. No. 4356170 and further details of which are presented in Example17. The mean molecular size of the PRP molecules used for conjugationwas determined as being approximately 20,000 Daltons. The conjugationwas carried out without a linker molecule but may also be carried outwith a linker molecule. A PRP/D15 molar ratio of approximately 7 wasused to provide an excess of PRP hapten.

The PRP/rD15 conjugate was tested according to the protocol of Example18 for immunogenicity in rabbits and elicited both primary and secondaryanti-PRP IgG and anti-D15 antibody responses (Table 9). Rabbitanti-rD15-PRP antisera also strongly reacted with both native D15 andrD15 as judged by immunoblot analysis. These data indicate that rD15 canbe used as a carrier protein in a conjugate vaccine. In addition, arD15-PRP conjugate vaccine should ensure a more consistent protectionagainst H. influenzae type b disease, particularly in infants, as aresult of the additional homotypic protection provided by antibodiesdirected against the D15 protein.

Example 15

This Example describes the preparation of D15 peptides.

D15 peptides (Table 2) were synthesized using an ABI 430A peptidesynthesizer and optimized t-Boc chemistry as described by themanufacturer, then cleaved from the resin by hydrof luoric acid (HF).The peptides were purified by reversed-phase high performance liquidchromatography (RP-HPLC) on a Vydac C4 semi-preparative column (1×30 cm)using a 15 to 55% acetonitrile gradient in 0.1% trifluoryl acetic acid(TFA) developed over 40 minutes at a flow rate of 2 mL/min. Allsynthetic peptides (Table 2) used in biochemical and immunologicalstudies were >95% pure as judged by analytical HPLC. Amino acidcomposition analyses of these peptides performed on a Waters Pico-Tagsystem were in good agreement with their theoretical compositions.

Example 16

This Example describes the protocol used for D15 peptide-specificantisera production.

Guinea pigs and rabbits were immunized with individual peptides (50 to200 μg) emulsified with Freund's complete adjuvant and injectedintramuscularly. After two booster doses with the same amount of peptidein incomplete Freund's adjuvant at +14 and +28 days, the anti-peptideantisera were collected on day +42 and tested by ELISAs andimmunoblotting. Both rabbit and guinea pig antisera were shown to bemonospecific for their respective immunizing peptides by thepeptide-specific ELISAs (Table 6). In addition, both guinea pig andrabbit antisera raised against D15 peptides reacted with both H.influenzae type b and non-typeable D15 on immunoblot analyses. Sincemost D15 peptides induced strong anti-peptide antibody responses in atleast one animal species, they are appropriate immunogens to be includedin immunogenic compositions including vaccine preparations.

Example 17

This Example describes the procedure used for the preparation of PRP-BSAconjugates.

0.5 mL of periodate-oxidized PRP (25 mg in 1 mL of 0.1 M sodiumphosphate buffer, pH 6.0), prepared from native PRP treated with aqueousperiodic acid (Carlone et al, 1986), was added to bovine serum albumin(BSA) (1.32 mg ; 0.02 μl) in 0.5 mL of 0.2 M sodium phosphate buffer, pH8.0, followed by the addition of sodium cyanoborohydride (14 μg ;0.22μmol ; 10 eqv. to BSA). After incubation at 37° C. for 5 days, thereaction mixture was dialysed against 4 L of 0.1 M phosphate buffer, pH7.5. The resulting solution was applied onto an analytical Superose 12column (15×300 mm, Pharmacia) equilibrated with 0.2 M sodium phosphatebuffer, pH 7.2, and eluted with the same buffer. Fractions weremonitored for absorbance at 230 nm. The first major protein peak waspooled and concentrated in a Centriprep 30 to 2.2 mL. The amount ofprotein was determined using the Bio Rad protein assay, and was found tobe 300 μg/mL. The presence of PRP in the protein conjugate fraction wasconfirmed by the Orcinol test.

Example 18

This Example describes the protocol used for the production of anti-PRPantisera in animals using rD15 conjugates.

Rabbits were immunized intramuscularly with rD15-PRP conjugates (Example14) (5 to 50 μg PRP equivalent) mixed with 3 mg AlPO₄ per mL, followedby two booster doses (half amount of the same immunogen) at 2 weekintervals. Antisera were collected every 2 weeks after the firstinjection, heat-inactivated at 56° C. for 30 minutes and stored at −20°C.

Example 19

This Example illustrates the reactivity between D15 peptides andanti-peptide and D15-specific antisera using D15-specific andpeptide-specific ELISAS.

Microtiter wells (Nunc-Immunoplate, Nunc, Denmark) were coated with 200ng of purified rD15 or 500 ng of individual peptides in 50 μL of coatingbuffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) for 16 hours at roomtemperature. The plates were then blocked with 0.1% (w/v) BSA inphosphate buffer saline (PBS) for 30 minutes at room temperature.Serially diluted antisera were added to the wells and incubated for 1hour at room temperature. After removal of the antisera, the plates werewashed five times with PBS containing 0.1% (w/v) Tween-20 and 0.1% (w/v)BSA. F(ab′)₂ fragments from goat anti-rabbit, guinea pig, mouse, orhuman IgG antibodies conjugated to horseradish peroxidase (JacksonImmunoResearch Labs Inc., PA) were diluted (1/8,000) with washingbuffer, and added onto the microtiter plates. After 1 hr incubation atroom temperature, the plates were washed five times with the washingbuffer. The plates were then developed using the substratetetramethylbenzidine (TMB) in H₂O₂ (ADI, Toronto). The reaction wasstopped with 1N H₂SO₄ and the optical density was measured at 450 nmusing a Titretek Multiskan II (Flow Labs., Virginia). Two irrelevantpeptides as negative controls in the peptide-specific ELISAs. Assayswere performed in triplicate, and the reactive titer of each antiserumwas defined as the dilution consistently showing 2-fold increaseabsorbance value over those obtained from the negative controls. Theresults obtained are summarized in Tables 3, 6 and 8 and in the DETAILEDDESCRIPTION OF THE INVENTION above.

Example 20

This Example illustrates the measurement of the anti-PRP IgG titers inrabbit anti-PRP-D15 conjugate antisera using a PRP-specific ELISA.

Microtiter wells (Nunc-Immunoplate, Nunc, Denmark) were coated with 200ng of purified PRP-BSA (see Example 17) in 200 μL of coating buffer (15mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) for 16 hours at room temperature. Theplates were then blocked with 0.1% (w/v) BSA in phosphate buffer saline(PBS) for 30 minutes at room temperature. Serially diluted rabbitantisera raised against PRP-D15 conjugates were added to the wells andincubated for 1 hour at room temperature. After removal of the antisera,the plates were washed five times with PBS containing 0.1% (w/v)Tween-20 and 0.1% (w/v) BSA. F(ab′)₂ fragment from goat anti-rabbit IgGantibodies conjugated to horseradish peroxidase (Jackson ImmunoResearchLabs Inc., PA) were diluted (1/8,000) with washing buffer, and addedonto the microtiter plates. After 1 hour incubation at room temperature,the plates were washed five times with the washing buffer. The plateswere then developed using the substrate tetramethylbenzidine (TMB) inH₂O₂ (ADI, Toronto). The reaction was stopped with 1N H₂SO₄ and theoptical density measured at 450 nm using a Titretek Multiskan II (FlowLabs., Virginia). A standard anti-PRP antiserum of known titer wasincluded as positive control. Assays were performed in triplicate, andthe reactive titer of each antiserum was defined as the reciprocal ofthe dilution consistently showing a 2-fold increase in O.D. value overthat obtained with the pre-immune serum (Table 9).

Example 21

This Example describes the protocol used for the production ofD15-specific antisera using purified D15, rD15 or N-terminal rD15fragment.

New Zealand White rabbits (Maple Lane) and guinea pigs (Charles River)were immunized intramuscularly (IM) with a 10 μg dose of eitheraffinity-purified native D15 (Example 13), recombinant D15 (Example 8)or N-terminal rD15 fragment (Example 11) emulsified in Freund's completeadjuvant (Difco). Animals were boosted on day 28 with another 10 μg doseof affinity-purified D15 or rD15 or rD15 fragment emulsified in Freund'sincomplete adjuvant and bled on day 42 via the marginal ear vein.D15-specific polyclonal antibodies were purified from this material asdescribed in Example 12.

Example 22

This Example illustrates the protective activity of D15-specificantisera against H. influenzae type b challenge using the infant ratmodel of bacteremia.

Five-day old infant rats were inoculated subcutaneously (SC) on thedorsum with 0.15 mL of two different rabbit anti-N-terminal rD15fragments. Pre-immune sera were used as negative controls. One day afterimmunization, the infant rats were injected intraperitoneally (IP) with200 colony-forming units (cfu) of Haemophilus influenzae type b Minn Astrain (0.1 ml) freshly grown in brain heart infusion (BHI) mediumsupplemented with cofactors and diluted in PBS containing 0.5 mM MgCl₂and 0.15 mM CaCl₂. One day later, blood samples were collected viacardiac puncture under methoxyflurane anaesthesia and plated onchocolate agar plates. The number of bacteria per mL of blood wasquantified after 24 hr. The statistical significance of differencesobserved in the levels of bacteremia relative to controls was analyzedby the Student's t-test. The results are summarized in Table 1.

Example 23

This Example describes the protocol used for the generation ofD15-specific T-cell lines.

BALB/c (H-2 ^(d)) mice purchased from Charles River Animal Farm(Montreal, Canada) were individually primed subcutaneously with 20 μg ofrD15 adsorbed to 1.5 mg of aluminium phosphate (alum). The animals wereboosted twice with the same dose of immunogen at 3 week intervals. Tendays after the final boost, spleens of immunized mice were removed.Splenocytes were cultured at 5.75×10⁵ cells per well in a final volumeof 200 μL of RPMI 1640 medium (Flow Lab.) supplemented with 10%heat-inactivated fetal calf serum (Gibco), 2 mM L-glutamine (Flow Lab.),100 U/mL) penicillin (Flow Lab.) and 5×10⁻⁵ M 2-mercaptoethanol (Sigma)in the presence of varying concentrations (1, 10 and 100 μg per mL) ofindividual D15 peptides (Table 2) in 96-well plates (Nunc, Denmark).Cultures were kept in a humidified incubator in the presence of 5%CO₂/air. Triplicate cultures were performed for each concentration ofeach peptide. Five days later, 150 μL of 10% rat concanavalin A culturesupernatant diluted in culture medium was added to the microtiter platewells as a source of Interleukin-2 (IL-2) to expand peptide-specificT-cells. Six days later, 150 μL of supernatant were removed from eachmicroculture, and 150 μL of fresh IL-2 containing culture supernatantadded to further expand and maintain the viability of thepeptide-specific T-cells. After a further 6 day-incubation, the cellswere washed three times, each time with 200 μL of culture medium.

Each set of cultures was then stimulated with the correspondingconcentrations (1, 10 and 100 μg per mL) of the peptide in the presenceof 2×10⁵ irradiated (1,500 rad) BALB/c spleen cells in a final volume of200 μL of culture medium. Sixty μL of supernatant were then removed fromeach microculture. The supernatants from each triplicate cultures setwere pooled. All supernatants were assayed for IL-2, Interleukin-4 andInterferon-gamma (IFN-γ). Detections of IL-2 and IL-4 were performedusing murine IL-2 and IL-4 ELISA kits purchased from Endogen Inc. (Ma,USA) respectively. Assay of IFN-γ was performed using a mouse IFN-γELISA kit supplied by Genzyme Corporation (Ma, USA). Test culturesupernatants were assayed at 1 in 5 dilution according to themanufacturers' instructions. The results obtained are set forth in Table7.

Example 25

This Example describes the general procedure used for the production ofmurine D15-specific monoclonal antibodies.

BALB/c mice were immunized intraperitoneally with 20 to 50 μg of theN-terminal rD15 fragment (Example 11) emulsified in Freund's completeadjuvant. Two weeks later, the mice were given another injection of thesame amount of immunogen in incomplete Freund's adjuvant (IFA). Threedays before the fusion, the mice were boosted again with the same amountof immunogen in IFA. Hybridomas were produced by fusion of spleniclymphocytes from immunized mice with non-secreting Sp2/0 myeloma cellsas previously described by Hamel et al. (1987). D15-specific hybridomaswere cloned by sequential limiting dilutions and screened for anti-D15monoclonal antibody production. Eight D15-specific hybridoma cell lineswere identified, expanded and frozen in liquid nitrogen. One of thehybridoma cell lines, 6C8-F6-C6, has been partially characterized. Themonoclonal antibody (MAb 6C8-F6-C6) reacts with peptide D15-P8. This MAb6C8-F6-C6 was used to prepare the D15-specific MAb affinity column andpurify native D15 from H. influenzae cell paste (Example 13).

TABLE 1 PROTECTIVE EFFECT OF PASSIVELY TRANSFERRED ANTI-N-TERMINAL RD15FRAGMENT ANTIBODIES IN THE INFANT RAT MODEL OF BACTEREMIA¹ cfu/0.1 mLblood Rabbit antisera Pre-immune Post-immunization p value Rb#434 510(6/6)² 6 (1/6) <0.001 Rb#435 910 (4/4)  6 (1/4) <0.001 ¹Five-day oldinfant rats were passively immunized with 0.15 mL of rabbitanti-N-terminal rD15 fragment s.c. One day later, the infant rats werechallenged with 200 cfu of H. influenzae type b strain MinnA (0.1 mL,IP). The blood samples were taken from each rat 24 hours after thechallenge and analysed for bacteria counts. ²The parentheses indicatethe number of rats found to be bacteremic out of the total number ofrats challenged.

TABLE 2 SEQUENCE OF OVERLAPPING SYNTHETIC PEPTIDES ENCOMPASSING THEENTIRE D15 ANTIGEN SEQUENCE PEPTIDES RESIDUES SEQUENCES SEQ ID NO:D15-P1 20-49 APFVAKDIRVDGVQGDLEQQIRASLPVRAG 14 D15-P2 45-74PVRAGQRVTDNDVAMIVRSLFVSGRFDDVK 15 D15-P3 68-99GRFDDVKAHQEGDVLVVSVVAKSIISDVKIKG 16 D15-P4  93-122SDVKIKGNSVIPTEALKQNLDANGFKVGDV 17 D15-P5 114-143ANGFKVGDVLIREKLNEFAKSVKEHYASVG 18 D15-P6 135-164VKEHYASVGRYNATVEPIVNTLPNNRAEIL 19 D15-P7 157-187PNNRAEILIQINEDDKAKLASLTFKGNESVS 20 D15-P8 180-209FKGNESVSSSTLQEQMELQPDSWWKKLWGNK 21 D15-P9 199-228PDSWWKLWGNKFEGAQFEKDLQSIRDYYLN 22 D15-P10 219-249LQSIRDYYLNNGYAKAQITKTDVQLNDEKTK 23 D15-P11 241-270VQLNDEKTKVNVTIDVNEGLQYDLRSARII 24 D15-P12 262-291YDLRSARIIGNLGGMSAELEPLLSALHLND 25 D15-P13 282-312PLLSALHLNDTFRRSDIADVENAIKAKLGER 26 D15-P14 304-333AIKAKLGERGYGSATVNSVPDFDDANKTLA 27 D15-P15 325-354FDDANKTLAITLVVDAGRRLTVRQLRFEGN 28 D15-P16 346-375VRQLRFEGNTVSADSTLRQEMRQQEGTWYN 29 D15-P17 367-396RQQEGTWYNSQLVELGKIRLDRTGFFETVE 30 D15-P18 390-416GFFETVENRIDPINGSNDEVDVVYKVK 31 D15-P19 410-435DVVYKVKERNTGSINFGIGYGTESGI 32 D15-P20 430-455 GTESGISYQASVKQDNFLGTGAAVSI33 D15-P21 450-477 GAAVSIAGTKNDYGTSVNLGYTEPYFTK 34 D15-P22 471-497TEPYFTKDGVSLGGNVFFENYDNSKSD 35 D15-P23 491-516YDNSKSDTSSNYKRTTYGSNVTLGFP 36 D15-P24 511-538VTLGFPVNENNSYYVGLGHTYNKISNF 37 D15-P25 532-559YNKISNFALEYNRNLYIQSMKFKGNGIK 38 D15-P26 554-582KGNGIKTNDFDFSFGWNYNSLNRGYFPTK 39 D15-P27 577-602GYFPTKGVKASLGGRVTIPGSDNKYYK 40 D15-P28 596-625SDNKYYKLSADVQGFYPLDRDHLWVVSAK 41 D15-P29 619-646LWVVSAKASAGYANGFGNKRLPFYQTYT 42 D15-P30 641-666FYQTYTAGGIGSLRGFAYGSIGPNAI 43 D15-P31 662-688GPNAIYAEYGNGSGTGTFKKISSDVIG 44 D15-P32 681-709KISSDVIGGNAIATASAELIVPTPFVSDK 45 D15-P33 705-731FVSDKSQNTVRTSLFVDAASVWNTKWK 46 D15-P34 725-750VWNTKWKSDKNGLESDVLKRLPDYGK 47 D15-P35 745-771LPDYGKSSRIRASTGVGFQWQSPIGPL 48 D15-P36 769-798GPLVFSYAKPIKKYENDDVEQFQFSIGGSF 49

TABLE 3 REACTIVITY OF RABBIT AND GUINEA PIG ANTI-N-TERMINAL rD15FRAGMENT ANTISERA WITH D15 SYNTHETIC PEPTIDES Reactive Titers Rabbitantisera Guinea pig antisera Peptides 3434 435 858 859 860 D15-P1 4001,600 6,400 6,400 6,400 D15-P2 1,600 <100 100 100 <100 D15-P3 400 <100100 <100 <100 D15-P4 25,600 6,400 <100 <100 <100 D15-P5 6,400 400 1,60025,600 400 D15-P6 1,600 6,400 400 6,400 6,400 D15-P7 6,400 1,600 25,60025,600 25,600 D15-P8 6,400 6,400 25,600 409,600 409,600 D15-P9 <100 <100400 1,600 1,600 D15-P10 <100 <100 400 6,400 <100

TABLE 4 INHIBITION OF ANTI-N-TERMINAL rD15 FRAGMENT ANTIBODY-INDUCEDPROTECTION BY D15 PEPTIDES IN THE INFANT RAT MODEL OF BACTEREMIA cfu ineach group/cfu in group #4 Group # Antibody cfu/10 μl blood (control)(%) 1 Anti-D15 Ab + PBS  60 ± 120 (3/7)  3 2 Anti-D15 Ab + 300 ± 240(6/7)  13 peptides 3 Anti-D15 Ab + 1,520 ± 1,280 (7/7)  64 rD15 4 PBS +peptides 2,360 ± 1,200 (6/7) 100 One half mL of rabbit anti-N-terminalrD15 fragment antiserum (Anti-rD15 fragment Ab) was mixed with eithernine D15 peptides (100 μg of peptides D15-P2 to D15-P10, See TABLE 2) orwith 600 μg of N-terminal rD15 fragment at room temperature for 1 hr.Antiserum and peptides mixed with PBS were used as controls. Seven-dayold infant rats were injected s.c. with 0.2 mL of the variouspreparations. After 24 h, the infant rats were challenged I.P. with 200cfu #of H. influenzae type b strain MinnA. The blood samples were takenat 24 h after the challenge. The numbers in parentheses indicate thenumber of animals that were bacteremic out of the total number ofanimals challenged. The level of bacteremia is expressed as the mean ofvalues obtained from seven infant rats tested individually ± onestandard deviation (SD).

TABLE 5 INHIBITION OF THE IMMUNOPROTECTION ABILITY OF THE RABBITANTI-N-TERMINAL rD15 FRAGMENT ANTISERUM ABSORBED WITH D15 PEPTIDES(D15-P4 TO D15-P8) IN THE INFANT RAT MODEL OF BACTEREMIA cfu in eachgroup/cfu Group # Antibody cfu/10 μl blood in group #3 (%) 1 rD15 Ab +PBS   220 ± 360 (3/6)  8 2 rD15 Ab + 2,960 ± 560 (6/6) 106 peptides 3PBS + peptides 2,800 ± 360 (6/6) 100 One half mL of rabbit anti-rD15fragment antiserum (rD15 Ab) was mixed with five D15 peptides (peptidesP4 to P8, 250 μg of each peptide) at room temperature for 1 hr.Antiserum and peptides diluted in PBS were used as controls. Seven-dayold infant rats were injected s.c. with 0.2 mL of the indicatedmaterial. After 24 h, the infant rats were challenged I.P. with 200 cfuof H. influenzae type b strain MinnA. The blood samples were collected24 h after challenge. The #numbers in parentheses indicate the number ofanimals that were bacteremia out of the total number of animalschallenged. The level of bacteremia is expressed as the mean of valuesobtained from six infant rats tested individually ± one SD.

TABLE 6 REACTIVITY OF RABBIT, GUINEA PIG AND MOUSE ANTI-rD15 ANTISERAWITH D15 PEPTIDES Reactive Titer¹ Peptide Rabbit² Guinea Pig³ Mouse⁴D15-P1  − − + D15-P2  − +++ + D15-P3  − − + D15-P4  + + + D15-P5  − − +D15-P6  − + + D15-P7  − − + D15-P8  − ++++ ++++ D15-P9  − − + D15-P10 −− +++ D15-P11 − − +++ D15-P12 − − + D15-P13 − − + D15-P14 +++ + +D15-P15 − − + D15-P16 − − + D15-P17 − − + D15-P18 − − + D15-P19 − − +D15-P20 − − + D15-P21 − − + D15-P22 − − + D15-P23 − − + D15-P24 − − +D15-P25 − − + D15-P26 − − +++ D15-P27 − + + D15-P28 − − + D15-P29 − − +D15-P30 − − + D15-P31 − − + D15-P32 − − + D15-P33 − − + D15-P34 − − +D15-P35 − − + D15-P36 ++++ − + ¹The reactive titer is based onpeptide-specific ELISAs. +, ++, +++, and ++++ represent reactive titersof animal antisera tested at 1/300, 1/1000, 1/2000, and 1/5000dilutions, respectively; − means nonreactive. ²Titer represents theaverage value of two rabbit antisera raised against rD15. ³Titerrepresents the average value of two guinea pig antisera raised againstrD15. ⁴Titer represents the average value of five mouse antisera raisedagainst r15.

TABLE 7 T-CELL STIMULATORY ACTIVITY OF D15 PEPTIDES CYTOKINE RELEASE(pg/mL)¹ Peptide IL-2² γ-IFN³ IL-4⁴ D15-P1  − − − D15-P2  122 − −D15-P3   25 − − D15-P4  − − − D15-P5  742 38,000  13 D15-P6  − − −D15-P7  − − − D15-P8  − − − D15-P9  − − − D15-P10 108 1,900 − D15-P11 −− − D15-P12 1,052   6,100 − D15-P13 105 6,200 56 D15-P14 − − − D15-P15 −− − D15-P16  48 − − D15-P17 − − − D15-P18  32 4,800 − D15-P19 88224,500  − D15-P20 − − − D15-P21 − − − D15-P22 − − − D15-P23  78 − −D15-P24 103 − − D15-P25 − − − D15-P26 572 6,700 − D15-P27 274 7,505 68D15-P28 142   742 − D15-P29 − − − D15-P30 − − − D15-P31 − − − D15-P32 −− − D15-P33 − − − D15-P34  82   603 − D15-P35 107   751 − D15-P36 − − −¹Results are expressed as mean values of triplicate cultures. Allstandard deviations were less than 15%. Immunodominant Th1-cell epitopesare highlighted with bold and Th0-cell epitopes are in italics.

TABLE 8 RABBIT AND GUINEA PIG ANTIBODY RESPONSES TO D15 PEPTIDESPeptide-specific ELISAs Reactive Titer¹ Immunogen Rabbit² Guinea Pig³D15-P1  102,400 819,200 D15-P2  204,800 1,637,400 D15-P3   51,2001,637,400 D15-P4  204,800 819,200 D15-P5   51,200 1,637,400 D15-P6  51,200 409,600 D15-P7  204,800 819,200 D15-P8   51,200 409,600 D15-P9 102,400 409,600 D15-P10 102,400 819,200 D15-P11  51,200 819,200 D15-P12102,400 204,800 D15-P13 NT⁴ 204,800 D15-P14 NT 409,600 D15-P15 NT204,800 D15-P16 NT 819,200 D15-P17 NT 204,800 D15-P18 NT 312,500 D15-P19NT 312,500 D15-P20 NT 62,500 D15-P21 NT 62,500 D15-P22 NT 12,500 D15-P23NT 1,562,500 D15-P24 NT 312,500 D15-P25 NT 62,500 D15-P26 NT 500 D15-P27NT 1,500 D15-P28 NT 1,250 D15-P29 NT <500 D15-P30 NT <500 D15-P31 NT<500 D15-P32 NT 12,500 D15-P33 NT 12,500 D15-P34 NT 62,500 D15-P35 NT1,250 D15-P36 NT 12,500 ¹The reactive titer is based on peptide-specificELISAs. A titer below 500 indicates that the peptide is not immunogenic.²Titers represent the average value of obtained for two rabbit antiseraraised against the D15 peptide. ³Titers represent the average valueobtained for two guinea pig antisera raised against the D15 peptide.⁴NT: not tested.

TABLE 9 RABBIT IgG ANTIBODY RESPONSE TO D15-PRP CONJUGATE Reactive TiterAgainst² PRP rD15 Rabbit¹ # 2 doses 3 doses 2 doses 3 doses 489-1 1,6003,200 1,600  6,400 490-1 1,600 1,600 6,400 25,600 ¹Rabbits wereimmunized intramuscularly with rD15-PRP conjugates (5 to 50 μg PRPequivalent) mixed with 3 mg ALPO₄ per mL, followed by two booster doses(half amount of the same immunogen) at 2 week intervals. ²Reactivetitres is based on PRP specific and D-15 specific ELISAs.

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the present invention provides purifiedand isolated nucleic acid molecules containing genes encoding the D15outer membrane protein, the sequences of these genes and the derivedamino acid sequences thereof. The invention also provides peptidescorresponding to portions of the D35 outer membrane protein. Inaddition, the invention provides antibodies raised against D15 outermembrane protein, fragments and peptides. The genes, DNA sequences,antibodies and peptides are useful for diagnosis, immunization and thegeneration of diagnostic and immunological reagents. Vaccines based onexpressed recombinant D35, portions thereof or peptides derived from theprovided sequences can be prepared for prevention of H. influenzaedisease. Modification are possible within the scope of the invention.

REFERENCES

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Ulman et al., 1993.

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Thomas W. R. and Rossi A. A. (1986) Infect. Immun. 52:812-817.

Thomas W. R. et al. (1990) Infect. and Immun. 58:1909-1913.

Carlone G. M. et al. (1986) J. Clin. Microbiol. 24:330-331.

Smith, D. B. and Johnson K. S. (1988) Gene 67:31-40.

Harkness, R. et al. (1992) J. Bacteriol. 174:2425-2430.

Hamel et al. (1987) J. Med. Microbiol. 23:163-170.

Mills et al. (1993) Infect. Immun. 61:399-410.

Trinchieri, (1993) Immunol. Today 14:335-338.

Hope, T. P. (1986) J. Immunol. Methods 88:1-18.

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Loeb et al. 1987. Infect. Immun. 55:2612-2618.

Panezutti, 1993. Infect. Immun. 61:1867-1872.

55 2949 base pairs nucleic acid single linear DNA (genomic) not providedCDS 75..2465 1 GATTACGCCA AGCTTAACGG TGTTTGCATT ATTTAATGAT TTTTTACGTCTATAATTTAT 60 ATAGGATACA ATCG ATG AAA AAA CTT CTA ATC GCA AGT TTA TTATTC GGT 110 Met Lys Lys Leu Leu Ile Ala Ser Leu Leu Phe Gly 1 5 10 ACGACA ACG ACT GTG TTT GCC GCA CCT TTT GTG GCA AAA GAT ATT CGT 158 Thr ThrThr Thr Val Phe Ala Ala Pro Phe Val Ala Lys Asp Ile Arg 15 20 25 GTG GATGGT GTT CAA GGT GAC TTA GAA CAA CAA ATC CGA GCA AGT TTA 206 Val Asp GlyVal Gln Gly Asp Leu Glu Gln Gln Ile Arg Ala Ser Leu 30 35 40 CCT GTT CGTGCC GGT CAG CGT GTG ACT GAC AAT GAT GTG GCT AAT ATT 254 Pro Val Arg AlaGly Gln Arg Val Thr Asp Asn Asp Val Ala Asn Ile 45 50 55 60 GTC CGC TCTTTA TTC GTA AGT GGT CGA TTC GAT GAT GTG AAA GCG CAT 302 Val Arg Ser LeuPhe Val Ser Gly Arg Phe Asp Asp Val Lys Ala His 65 70 75 CAA GAA GGC GATGTG CTT GTT GTT AGC GTT GTG GCT AAA TCG ATC ATT 350 Gln Glu Gly Asp ValLeu Val Val Ser Val Val Ala Lys Ser Ile Ile 80 85 90 TCA GAT GTT AAA ATCAAA GGT AAC TCT GTT ATT CCC ACT GAA GCA CTT 398 Ser Asp Val Lys Ile LysGly Asn Ser Val Ile Pro Thr Glu Ala Leu 95 100 105 AAA CAA AAC TTA GATGCT AAC GGG TTT AAA GTT GGC GAT GTT TTA ATT 446 Lys Gln Asn Leu Asp AlaAsn Gly Phe Lys Val Gly Asp Val Leu Ile 110 115 120 CGA GAA AAA TTA AATGAA TTT GCC AAA AGT GTA AAA GAG CAC TAT GCA 494 Arg Glu Lys Leu Asn GluPhe Ala Lys Ser Val Lys Glu His Tyr Ala 125 130 135 140 AGT GTA GGT CGCTAT AAC GCA ACA GTT GAA CCT ATT GTC AAT ACG CTA 542 Ser Val Gly Arg TyrAsn Ala Thr Val Glu Pro Ile Val Asn Thr Leu 145 150 155 CCA AAT AAT CGCGCT GAA ATT TTA ATT CAA ATC AAT GAA GAT GAT AAA 590 Pro Asn Asn Arg AlaGlu Ile Leu Ile Gln Ile Asn Glu Asp Asp Lys 160 165 170 GCA AAA TTG GCATCA TTA ACT TTC AAG GGG AAC GAA TCT GTT AGT AGC 638 Ala Lys Leu Ala SerLeu Thr Phe Lys Gly Asn Glu Ser Val Ser Ser 175 180 185 AGT ACA TTA CAAGAA CAA ATG GAA TTA CAA CCT GAT TCT TGG TGG AAA 686 Ser Thr Leu Gln GluGln Met Glu Leu Gln Pro Asp Ser Trp Trp Lys 190 195 200 TTA TGG GGA AATAAA TTT GAA GGT GCG CAA TTC GAG AAA GAT TTG CAG 734 Leu Trp Gly Asn LysPhe Glu Gly Ala Gln Phe Glu Lys Asp Leu Gln 205 210 215 220 TCA ATT CGTGAT TAT TAT TTA AAT AAT GGC TAT GCC AAA GCA CAA ATT 782 Ser Ile Arg AspTyr Tyr Leu Asn Asn Gly Tyr Ala Lys Ala Gln Ile 225 230 235 ACT AAA ACGGAT GTT CAG CTA AAT GAT GAA AAA ACA AAA GTT AAT GTA 830 Thr Lys Thr AspVal Gln Leu Asn Asp Glu Lys Thr Lys Val Asn Val 240 245 250 ACC ATT GATGTA AAT GAA GGT TTA CAG TAT GAC CTT CGT AGT GCA CGC 878 Thr Ile Asp ValAsn Glu Gly Leu Gln Tyr Asp Leu Arg Ser Ala Arg 255 260 265 ATT ATA GGTAAT CTG GGA GGT ATG TCT GCC GAG CTT GAA CCT TTA CTT 926 Ile Ile Gly AsnLeu Gly Gly Met Ser Ala Glu Leu Glu Pro Leu Leu 270 275 280 TCA GCA TTACAT TTA AAT GAT ACT TTC CGC CGT AGT GAT ATT GCA GAT 974 Ser Ala Leu HisLeu Asn Asp Thr Phe Arg Arg Ser Asp Ile Ala Asp 285 290 295 300 GTA GAAAAT GCA ATT AAA GCA AAA CTT GGA GAA CGC GGT TAC GGT AGC 1022 Val Glu AsnAla Ile Lys Ala Lys Leu Gly Glu Arg Gly Tyr Gly Ser 305 310 315 GCA ACGGTA AAT TCA GTA CCT GAT TTT GAT GAT GCA AAT AAA ACA TTA 1070 Ala Thr ValAsn Ser Val Pro Asp Phe Asp Asp Ala Asn Lys Thr Leu 320 325 330 GCG ATAACC CTT GTT GTT GAT GCT GGA CGA CGT TTA ACT GTT CGC CAA 1118 Ala Ile ThrLeu Val Val Asp Ala Gly Arg Arg Leu Thr Val Arg Gln 335 340 345 CTT CGCTTT GAA GGA AAT ACC GTT TCT GCT GAT AGC ACT TTA CGT CAG 1166 Leu Arg PheGlu Gly Asn Thr Val Ser Ala Asp Ser Thr Leu Arg Gln 350 355 360 GAA ATGCGC CAA CAA GAA GGA ACT TGG TAT AAT TCA CAA TTA GTT GAG 1214 Glu Met ArgGln Gln Glu Gly Thr Trp Tyr Asn Ser Gln Leu Val Glu 365 370 375 380 TTAGGA AAA ATT CGC TTA GAT CGT ACA GGT TTC TTC GAA ACA GTC GAA 1262 Leu GlyLys Ile Arg Leu Asp Arg Thr Gly Phe Phe Glu Thr Val Glu 385 390 395 AACCGA ATT GAT CCT ATC AAT GGT AGT AAT GAT GAA GTG GAT GTC GTA 1310 Asn ArgIle Asp Pro Ile Asn Gly Ser Asn Asp Glu Val Asp Val Val 400 405 410 TATAAA GTC AAA GAA CGT AAC ACG GGT AGT ATC AAC TTT GGT ATT GGT 1358 Tyr LysVal Lys Glu Arg Asn Thr Gly Ser Ile Asn Phe Gly Ile Gly 415 420 425 TACGGT ACA GAG AGT GGT ATT AGT TAT CAA GCA AGT GTT AAA CAA GAT 1406 Tyr GlyThr Glu Ser Gly Ile Ser Tyr Gln Ala Ser Val Lys Gln Asp 430 435 440 AATTTC TTG GGA ACA GGG GCG GCA GTA AGT ATA GCT GGT ACG AAA AAT 1454 Asn PheLeu Gly Thr Gly Ala Ala Val Ser Ile Ala Gly Thr Lys Asn 445 450 455 460GAT TAT GGT ACG AGT GTC AAT TTG GGT TAT ACC GAG CCC TAT TTT ACT 1502 AspTyr Gly Thr Ser Val Asn Leu Gly Tyr Thr Glu Pro Tyr Phe Thr 465 470 475AAA GAT GGT GTA AGT CTT GGT GGA AAT GTT TTC TTT GAA AAC TAC GAT 1550 LysAsp Gly Val Ser Leu Gly Gly Asn Val Phe Phe Glu Asn Tyr Asp 480 485 490AAC TCT AAA AGT GAT ACA TCC TCT AAC TAT AAG CGT ACG ACT TAC GGA 1598 AsnSer Lys Ser Asp Thr Ser Ser Asn Tyr Lys Arg Thr Thr Tyr Gly 495 500 505AGT AAT GTT ACT TTA GGT TTC CCT GTA AAT GAA AAT AAC TCC TAT TAT 1646 SerAsn Val Thr Leu Gly Phe Pro Val Asn Glu Asn Asn Ser Tyr Tyr 510 515 520GTA GGA TTA GGT CAT ACC TAT AAT AAA ATT AGT AAC TTT GCT CTA GAA 1694 ValGly Leu Gly His Thr Tyr Asn Lys Ile Ser Asn Phe Ala Leu Glu 525 530 535540 TAT AAC CGT AAT TTA TAT ATT CAA TCA ATG AAA TTT AAA GGT AAT GGC 1742Tyr Asn Arg Asn Leu Tyr Ile Gln Ser Met Lys Phe Lys Gly Asn Gly 545 550555 ATT AAA ACA AAT GAC TTT GAT TTT TCT TTT GGT TGG AAC TAT AAC AGC 1790Ile Lys Thr Asn Asp Phe Asp Phe Ser Phe Gly Trp Asn Tyr Asn Ser 560 565570 CTT AAT AGA GGC TAT TTC CCA ACT AAA GGG GTT AAA GCA AGT CTT GGT 1838Leu Asn Arg Gly Tyr Phe Pro Thr Lys Gly Val Lys Ala Ser Leu Gly 575 580585 GGA CGA GTT ACT ATT CCA GGT TCT GAT AAC AAA TAC TAC AAA CTA AGT 1886Gly Arg Val Thr Ile Pro Gly Ser Asp Asn Lys Tyr Tyr Lys Leu Ser 590 595600 GCA GAT GTA CAG GGT TTC TAC CCA TTA GAC AGA GAT CAC CTC TGG GTT 1934Ala Asp Val Gln Gly Phe Tyr Pro Leu Asp Arg Asp His Leu Trp Val 605 610615 620 GTA TCT GCA AAA GCA TCT GCA GGA TAT GCA AAT GGT TTT GGA AAC AAG1982 Val Ser Ala Lys Ala Ser Ala Gly Tyr Ala Asn Gly Phe Gly Asn Lys 625630 635 CGT TTA CCG TTC TAT CAA ACT TAT ACA GCG GGT GGC ATC GGT TCA TTA2030 Arg Leu Pro Phe Tyr Gln Thr Tyr Thr Ala Gly Gly Ile Gly Ser Leu 640645 650 CGT GGT TTT GCT TAT GGT AGT ATT GGA CCT AAC GCA ATT TAT GCC GAA2078 Arg Gly Phe Ala Tyr Gly Ser Ile Gly Pro Asn Ala Ile Tyr Ala Glu 655660 665 TAT GGT AAT GGT AGT GGT ACT GGT ACT TTT AAG AAG ATA AGT TCT GAT2126 Tyr Gly Asn Gly Ser Gly Thr Gly Thr Phe Lys Lys Ile Ser Ser Asp 670675 680 GTG ATT GGT GGT AAT GCA ATC GCT ACA GCT AGC GCA GAG TTA ATT GTG2174 Val Ile Gly Gly Asn Ala Ile Ala Thr Ala Ser Ala Glu Leu Ile Val 685690 695 700 CCA ACT CCA TTT GTG AGC GAT AAG AGC CAA AAT ACG GTC CGA ACCTCC 2222 Pro Thr Pro Phe Val Ser Asp Lys Ser Gln Asn Thr Val Arg Thr Ser705 710 715 TTA TTT GTT GAT GCG GCA AGT GTT TGG AAT ACT AAA TGG AAA TCAGAT 2270 Leu Phe Val Asp Ala Ala Ser Val Trp Asn Thr Lys Trp Lys Ser Asp720 725 730 AAA AAT GGA TTA GAG AGC GAT GTA TTA AAA AGA TTG CCT GAT TATGGC 2318 Lys Asn Gly Leu Glu Ser Asp Val Leu Lys Arg Leu Pro Asp Tyr Gly735 740 745 AAA TCA AGC CGT ATT CGC GCC TCT ACA GGT GTC GGA TTC CAA TGGCAA 2366 Lys Ser Ser Arg Ile Arg Ala Ser Thr Gly Val Gly Phe Gln Trp Gln750 755 760 TCT CCT ATT GGG CCA TTG GTA TTC TCT TAT GCC AAA CCA ATT AAAAAA 2414 Ser Pro Ile Gly Pro Leu Val Phe Ser Tyr Ala Lys Pro Ile Lys Lys765 770 775 780 TAT GAA AAT GAT GAT GTC GAA CAG TTC CAA TTT AGT ATT GGAGGT TCT 2462 Tyr Glu Asn Asp Asp Val Glu Gln Phe Gln Phe Ser Ile Gly GlySer 785 790 795 TTC TAATAAATTG AACTTTTTTC TTCATCAGAA CTCAAAAACAACGTTCTCTG 2515 Phe CCTAATTTAA TTGGGCAGAG AAAATATTAA ACCCATCATTTAATTAAGGA TATTTATCAA 2575 ATGAAAAACA TCGCAAAAGT AACCGCACTT GCTTTAGGTATTGCACTTGC TTCAGGCTAT 2635 GCTTCCGCTG AAGAAAAAAT TGCTTTCATT AATGCAGGTATATTTTTCAA CATCACCCAG 2695 ATCGCCAAGC GGTAGCAGAT AAACTTGATG CTGAATTTAAACCTGTAGCT GAGAAATTAG 2755 CAGCAAGCAA AAAAGAAGTT GATGATAAAA TTGCTGCTGCTCGTAAAAAA GTAGAAGCAA 2815 AAGTTGCGGC TTTAGAAAAA GATGCACCTC GCTTACGTCAAGCTGATATT CAAAAACGCC 2875 AACAGGAGAT TAATAAATTA GGTGCGGCTG AAGATGCTGAATTACAAAAA TTAATGCAAG 2935 AACAAGATAA AAAA 2949 797 amino acids aminoacid linear protein not provided 2 Met Lys Lys Leu Leu Ile Ala Ser LeuLeu Phe Gly Thr Thr Thr Thr 1 5 10 15 Val Phe Ala Ala Pro Phe Val AlaLys Asp Ile Arg Val Asp Gly Val 20 25 30 Gln Gly Asp Leu Glu Gln Gln IleArg Ala Ser Leu Pro Val Arg Ala 35 40 45 Gly Gln Arg Val Thr Asp Asn AspVal Ala Asn Ile Val Arg Ser Leu 50 55 60 Phe Val Ser Gly Arg Phe Asp AspVal Lys Ala His Gln Glu Gly Asp 65 70 75 80 Val Leu Val Val Ser Val ValAla Lys Ser Ile Ile Ser Asp Val Lys 85 90 95 Ile Lys Gly Asn Ser Val IlePro Thr Glu Ala Leu Lys Gln Asn Leu 100 105 110 Asp Ala Asn Gly Phe LysVal Gly Asp Val Leu Ile Arg Glu Lys Leu 115 120 125 Asn Glu Phe Ala LysSer Val Lys Glu His Tyr Ala Ser Val Gly Arg 130 135 140 Tyr Asn Ala ThrVal Glu Pro Ile Val Asn Thr Leu Pro Asn Asn Arg 145 150 155 160 Ala GluIle Leu Ile Gln Ile Asn Glu Asp Asp Lys Ala Lys Leu Ala 165 170 175 SerLeu Thr Phe Lys Gly Asn Glu Ser Val Ser Ser Ser Thr Leu Gln 180 185 190Glu Gln Met Glu Leu Gln Pro Asp Ser Trp Trp Lys Leu Trp Gly Asn 195 200205 Lys Phe Glu Gly Ala Gln Phe Glu Lys Asp Leu Gln Ser Ile Arg Asp 210215 220 Tyr Tyr Leu Asn Asn Gly Tyr Ala Lys Ala Gln Ile Thr Lys Thr Asp225 230 235 240 Val Gln Leu Asn Asp Glu Lys Thr Lys Val Asn Val Thr IleAsp Val 245 250 255 Asn Glu Gly Leu Gln Tyr Asp Leu Arg Ser Ala Arg IleIle Gly Asn 260 265 270 Leu Gly Gly Met Ser Ala Glu Leu Glu Pro Leu LeuSer Ala Leu His 275 280 285 Leu Asn Asp Thr Phe Arg Arg Ser Asp Ile AlaAsp Val Glu Asn Ala 290 295 300 Ile Lys Ala Lys Leu Gly Glu Arg Gly TyrGly Ser Ala Thr Val Asn 305 310 315 320 Ser Val Pro Asp Phe Asp Asp AlaAsn Lys Thr Leu Ala Ile Thr Leu 325 330 335 Val Val Asp Ala Gly Arg ArgLeu Thr Val Arg Gln Leu Arg Phe Glu 340 345 350 Gly Asn Thr Val Ser AlaAsp Ser Thr Leu Arg Gln Glu Met Arg Gln 355 360 365 Gln Glu Gly Thr TrpTyr Asn Ser Gln Leu Val Glu Leu Gly Lys Ile 370 375 380 Arg Leu Asp ArgThr Gly Phe Phe Glu Thr Val Glu Asn Arg Ile Asp 385 390 395 400 Pro IleAsn Gly Ser Asn Asp Glu Val Asp Val Val Tyr Lys Val Lys 405 410 415 GluArg Asn Thr Gly Ser Ile Asn Phe Gly Ile Gly Tyr Gly Thr Glu 420 425 430Ser Gly Ile Ser Tyr Gln Ala Ser Val Lys Gln Asp Asn Phe Leu Gly 435 440445 Thr Gly Ala Ala Val Ser Ile Ala Gly Thr Lys Asn Asp Tyr Gly Thr 450455 460 Ser Val Asn Leu Gly Tyr Thr Glu Pro Tyr Phe Thr Lys Asp Gly Val465 470 475 480 Ser Leu Gly Gly Asn Val Phe Phe Glu Asn Tyr Asp Asn SerLys Ser 485 490 495 Asp Thr Ser Ser Asn Tyr Lys Arg Thr Thr Tyr Gly SerAsn Val Thr 500 505 510 Leu Gly Phe Pro Val Asn Glu Asn Asn Ser Tyr TyrVal Gly Leu Gly 515 520 525 His Thr Tyr Asn Lys Ile Ser Asn Phe Ala LeuGlu Tyr Asn Arg Asn 530 535 540 Leu Tyr Ile Gln Ser Met Lys Phe Lys GlyAsn Gly Ile Lys Thr Asn 545 550 555 560 Asp Phe Asp Phe Ser Phe Gly TrpAsn Tyr Asn Ser Leu Asn Arg Gly 565 570 575 Tyr Phe Pro Thr Lys Gly ValLys Ala Ser Leu Gly Gly Arg Val Thr 580 585 590 Ile Pro Gly Ser Asp AsnLys Tyr Tyr Lys Leu Ser Ala Asp Val Gln 595 600 605 Gly Phe Tyr Pro LeuAsp Arg Asp His Leu Trp Val Val Ser Ala Lys 610 615 620 Ala Ser Ala GlyTyr Ala Asn Gly Phe Gly Asn Lys Arg Leu Pro Phe 625 630 635 640 Tyr GlnThr Tyr Thr Ala Gly Gly Ile Gly Ser Leu Arg Gly Phe Ala 645 650 655 TyrGly Ser Ile Gly Pro Asn Ala Ile Tyr Ala Glu Tyr Gly Asn Gly 660 665 670Ser Gly Thr Gly Thr Phe Lys Lys Ile Ser Ser Asp Val Ile Gly Gly 675 680685 Asn Ala Ile Ala Thr Ala Ser Ala Glu Leu Ile Val Pro Thr Pro Phe 690695 700 Val Ser Asp Lys Ser Gln Asn Thr Val Arg Thr Ser Leu Phe Val Asp705 710 715 720 Ala Ala Ser Val Trp Asn Thr Lys Trp Lys Ser Asp Lys AsnGly Leu 725 730 735 Glu Ser Asp Val Leu Lys Arg Leu Pro Asp Tyr Gly LysSer Ser Arg 740 745 750 Ile Arg Ala Ser Thr Gly Val Gly Phe Gln Trp GlnSer Pro Ile Gly 755 760 765 Pro Leu Val Phe Ser Tyr Ala Lys Pro Ile LysLys Tyr Glu Asn Asp 770 775 780 Asp Val Glu Gln Phe Gln Phe Ser Ile GlyGly Ser Phe 785 790 795 2984 base pairs nucleic acid single linear DNA(genomic) not provided CDS 374..2764 3 ACAGGACAGC TTTCCCTTTT AACCTTGAAAATATTAGGGA AATTACTTCC TGGCGATTTG 60 TCATTAAATA ATTTAAGTGG GCCAATTTCTATTGCAAAAG GTGCTGGCCC ATCAGCAAAT 120 ATTGGATTGG TGTATTTTTT AAGTTTTATGGCACTGATTA GTGTAAATTT AGGGATTATG 180 AATTTATTTC CATTACCAGT ATTAGATGGCGGTCATTTAG TTTTTTTAAC AATGGAAGCT 240 GTTAAAGGAA AACCTGTTTC TGAGCGGGTGCAAAGCATCT GTTATCGAAT TGGCGCAGCA 300 CTGTTATTAA GCTTAACGGT GTTTGCATTATTTAATGATT TTTTACGTCT ATAATTTATA 360 TAGGATACAA TCG ATG AAA AAA CTT CTAATC GCA AGT TTA TTA TTC GGT 409 Met Lys Lys Leu Leu Ile Ala Ser Leu LeuPhe Gly 1 5 10 ACG ACA ACG ACT GTG TTT GCC GCA CCT TTT GTG GCA AAA GATATT CGT 457 Thr Thr Thr Thr Val Phe Ala Ala Pro Phe Val Ala Lys Asp IleArg 15 20 25 GTG GAT GGT GTT CAA GGT GAC TTA GAA CAA CAA ATC CGA GCA AGTTTA 505 Val Asp Gly Val Gln Gly Asp Leu Glu Gln Gln Ile Arg Ala Ser Leu30 35 40 CCT GTT CGT GCC GGT CAG CGT GTG ACT GAC AAT GAT GTG GCT AAT ATT553 Pro Val Arg Ala Gly Gln Arg Val Thr Asp Asn Asp Val Ala Asn Ile 4550 55 60 GTC CGC TCT TTA TTC GTA AGT GGT CGA TTC GAT GAT GTG AAA GCG CAT601 Val Arg Ser Leu Phe Val Ser Gly Arg Phe Asp Asp Val Lys Ala His 6570 75 CAA GAA GGC GAT GTG CTT GTT GTT AGC GTT GTG GCT AAA TCG ATC ATT649 Gln Glu Gly Asp Val Leu Val Val Ser Val Val Ala Lys Ser Ile Ile 8085 90 TCA GAT GTT AAA ATC AAA GGT AAC TCT GTT ATT CCC ACT GAA GCA CTT697 Ser Asp Val Lys Ile Lys Gly Asn Ser Val Ile Pro Thr Glu Ala Leu 95100 105 AAA CAA AAC TTA GAT GCT AAC GGG TTT AAA GTT GGC GAT GTT TTA ATT745 Lys Gln Asn Leu Asp Ala Asn Gly Phe Lys Val Gly Asp Val Leu Ile 110115 120 CGA GAA AAA TTA AAT GAA TTT GCC AAA AGT GTA AAA GAG CAC TAT GCA793 Arg Glu Lys Leu Asn Glu Phe Ala Lys Ser Val Lys Glu His Tyr Ala 125130 135 140 AGT GTA GGT CGC TAT AAC GCA ACA GTT GAA CCT ATT GTC AAT ACGCTA 841 Ser Val Gly Arg Tyr Asn Ala Thr Val Glu Pro Ile Val Asn Thr Leu145 150 155 CCA AAT AAT CGC GCT GAA ATT TTA ATT CAA ATC AAT GAA GAT GATAAA 889 Pro Asn Asn Arg Ala Glu Ile Leu Ile Gln Ile Asn Glu Asp Asp Lys160 165 170 GCA AAA TTG GCA TCA TTA ACT TTC AAG GGG AAC GAA TCT GTT AGTAGC 937 Ala Lys Leu Ala Ser Leu Thr Phe Lys Gly Asn Glu Ser Val Ser Ser175 180 185 AGT ACA TTA CAA GAA CAA ATG GAA TTA CAA CCT GAT TCT TGG TGGAAA 985 Ser Thr Leu Gln Glu Gln Met Glu Leu Gln Pro Asp Ser Trp Trp Lys190 195 200 TTA TGG GGA AAT AAA TTT GAA GGT GCG CAA TTC GAG AAA GAT TTGCAG 1033 Leu Trp Gly Asn Lys Phe Glu Gly Ala Gln Phe Glu Lys Asp Leu Gln205 210 215 220 TCA ATT CGT GAT TAT TAT TTA AAT AAT GGC TAT GCC AAA GCACAA ATT 1081 Ser Ile Arg Asp Tyr Tyr Leu Asn Asn Gly Tyr Ala Lys Ala GlnIle 225 230 235 ACT AAA ACG GAT GTT CAG CTA AAT GAT GAA AAA ACA AAA GTTAAT GTA 1129 Thr Lys Thr Asp Val Gln Leu Asn Asp Glu Lys Thr Lys Val AsnVal 240 245 250 ACC ATT GAT GTA AAT GAA GGT TTA CAG TAT GAC CTT CGT AGTGCA CGC 1177 Thr Ile Asp Val Asn Glu Gly Leu Gln Tyr Asp Leu Arg Ser AlaArg 255 260 265 ATT ATA GGT AAT CTG GGA GGT ATG TCT GCC GAG CTT GAA CCTTTA CTT 1225 Ile Ile Gly Asn Leu Gly Gly Met Ser Ala Glu Leu Glu Pro LeuLeu 270 275 280 TCA GCA TTA CAT TTA AAT GAT ACT TTC CGC CGT AGT GAT ATTGCA GAT 1273 Ser Ala Leu His Leu Asn Asp Thr Phe Arg Arg Ser Asp Ile AlaAsp 285 290 295 300 GTA GAA AAT GCA ATT AAA GCA AAA CTT GGA GAA CGC GGTTAC GGT AGC 1321 Val Glu Asn Ala Ile Lys Ala Lys Leu Gly Glu Arg Gly TyrGly Ser 305 310 315 GCA ACG GTA AAT TCA GTA CCT GAT TTT GAT GAT GCA AATAAA ACA TTA 1369 Ala Thr Val Asn Ser Val Pro Asp Phe Asp Asp Ala Asn LysThr Leu 320 325 330 GCG ATA ACC CTT GTT GTT GAT GCT GGA CGA CGT TTA ACTGTT CGC CAA 1417 Ala Ile Thr Leu Val Val Asp Ala Gly Arg Arg Leu Thr ValArg Gln 335 340 345 CTT CGC TTT GAA GGA AAT ACC GTT TCT GCT GAT AGC ACTTTA CGT CAG 1465 Leu Arg Phe Glu Gly Asn Thr Val Ser Ala Asp Ser Thr LeuArg Gln 350 355 360 GAA ATG CGC CAA CAA GAA GGA ACT TGG TAT AAT TCA CAATTA GTT GAG 1513 Glu Met Arg Gln Gln Glu Gly Thr Trp Tyr Asn Ser Gln LeuVal Glu 365 370 375 380 TTA GGA AAA ATT CGC TTA GAT CGT ACA GGT TTC TTCGAA ACA GTC GAA 1561 Leu Gly Lys Ile Arg Leu Asp Arg Thr Gly Phe Phe GluThr Val Glu 385 390 395 AAC CGA ATT GAT CCT ATC AAT GGT AGT AAT GAT GAAGTG GAT GTC GTA 1609 Asn Arg Ile Asp Pro Ile Asn Gly Ser Asn Asp Glu ValAsp Val Val 400 405 410 TAT AAA GTC AAA GAA CGT AAC ACG GGT AGT ATC AACTTT GGT ATT GGT 1657 Tyr Lys Val Lys Glu Arg Asn Thr Gly Ser Ile Asn PheGly Ile Gly 415 420 425 TAC GGT ACA GAG AGT GGT ATT AGT TAT CAA GCA AGTGTT AAA CAA GAT 1705 Tyr Gly Thr Glu Ser Gly Ile Ser Tyr Gln Ala Ser ValLys Gln Asp 430 435 440 AAT TTC TTG GGA ACA GGG GCG GCA GTA AGT ATA GCTGGT ACG AAA AAT 1753 Asn Phe Leu Gly Thr Gly Ala Ala Val Ser Ile Ala GlyThr Lys Asn 445 450 455 460 GAT TAT GGT ACG AGT GTC AAT TTG GGT TAT ACCGAG CCC TAT TTT ACT 1801 Asp Tyr Gly Thr Ser Val Asn Leu Gly Tyr Thr GluPro Tyr Phe Thr 465 470 475 AAA GAT GGT GTA AGT CTT GGT GGA AAT GTT TTCTTT GAA AAC TAC GAT 1849 Lys Asp Gly Val Ser Leu Gly Gly Asn Val Phe PheGlu Asn Tyr Asp 480 485 490 AAC TCT AAA AGT GAT ACA TCC TCT AAC TAT AAGCGT ACG ACT TAC GGA 1897 Asn Ser Lys Ser Asp Thr Ser Ser Asn Tyr Lys ArgThr Thr Tyr Gly 495 500 505 AGT AAT GTT ACT TTA GGT TTC CCT GTA AAT GAAAAT AAC TCC TAT TAT 1945 Ser Asn Val Thr Leu Gly Phe Pro Val Asn Glu AsnAsn Ser Tyr Tyr 510 515 520 GTA GGA TTA GGT CAT ACC TAT AAT AAA ATT AGTAAC TTT GCT CTA GAA 1993 Val Gly Leu Gly His Thr Tyr Asn Lys Ile Ser AsnPhe Ala Leu Glu 525 530 535 540 TAT AAC CGT AAT TTA TAT ATT CAA TCA ATGAAA TTT AAA GGT AAT GGC 2041 Tyr Asn Arg Asn Leu Tyr Ile Gln Ser Met LysPhe Lys Gly Asn Gly 545 550 555 ATT AAA ACA AAT GAC TTT GAT TTT TCT TTTGGT TGG AAC TAT AAC AGC 2089 Ile Lys Thr Asn Asp Phe Asp Phe Ser Phe GlyTrp Asn Tyr Asn Ser 560 565 570 CTT AAT AGA GGC TAT TTC CCA ACT AAA GGGGTT AAA GCA AGT CTT GGT 2137 Leu Asn Arg Gly Tyr Phe Pro Thr Lys Gly ValLys Ala Ser Leu Gly 575 580 585 GGA CGA GTT ACT ATT CCA GGT TCT GAT AACAAA TAC TAC AAA CTA AGT 2185 Gly Arg Val Thr Ile Pro Gly Ser Asp Asn LysTyr Tyr Lys Leu Ser 590 595 600 GCA GAT GTA CAG GGT TTC TAC CCA TTA GACAGA GAT CAC CTC TGG GTT 2233 Ala Asp Val Gln Gly Phe Tyr Pro Leu Asp ArgAsp His Leu Trp Val 605 610 615 620 GTA TCT GCA AAA GCA TCT GCA GGA TATGCA AAT GGT TTT GGA AAC AAG 2281 Val Ser Ala Lys Ala Ser Ala Gly Tyr AlaAsn Gly Phe Gly Asn Lys 625 630 635 CGT TTA CCG TTC TAT CAA ACT TAT ACAGCG GGT GGC ATC GGT TCA TTA 2329 Arg Leu Pro Phe Tyr Gln Thr Tyr Thr AlaGly Gly Ile Gly Ser Leu 640 645 650 CGT GGT TTT GCT TAT GGT AGT ATT GGACCT AAC GCA ATT TAT GCC GAA 2377 Arg Gly Phe Ala Tyr Gly Ser Ile Gly ProAsn Ala Ile Tyr Ala Glu 655 660 665 TAT GGT AAT GGT AGT GGT ACT GGT ACTTTT AAG AAG ATA AGT TCT GAT 2425 Tyr Gly Asn Gly Ser Gly Thr Gly Thr PheLys Lys Ile Ser Ser Asp 670 675 680 GTG ATT GGT GGT AAT GCA ATC GCT ACAGCT AGC GCA GAG TTA ATT GTG 2473 Val Ile Gly Gly Asn Ala Ile Ala Thr AlaSer Ala Glu Leu Ile Val 685 690 695 700 CCA ACT CCA TTT GTG AGC GAT AAGAGC CAA AAT ACG GTC CGA ACC TCC 2521 Pro Thr Pro Phe Val Ser Asp Lys SerGln Asn Thr Val Arg Thr Ser 705 710 715 TTA TTT GTT GAT GCG GCA AGT GTTTGG AAT ACT AAA TGG AAA TCA GAT 2569 Leu Phe Val Asp Ala Ala Ser Val TrpAsn Thr Lys Trp Lys Ser Asp 720 725 730 AAA AAT GGA TTA GAG AGC GAT GTATTA AAA AGA TTG CCT GAT TAT GGC 2617 Lys Asn Gly Leu Glu Ser Asp Val LeuLys Arg Leu Pro Asp Tyr Gly 735 740 745 AAA TCA AGC CGT ATT CGC GCC TCTACA GGT GTC GGA TTC CAA TGG CAA 2665 Lys Ser Ser Arg Ile Arg Ala Ser ThrGly Val Gly Phe Gln Trp Gln 750 755 760 TCT CCT ATT GGG CCA TTG GTA TTCTCT TAT GCC AAA CCA ATT AAA AAA 2713 Ser Pro Ile Gly Pro Leu Val Phe SerTyr Ala Lys Pro Ile Lys Lys 765 770 775 780 TAT GAA AAT GAT GAT GTC GAACAG TTC CAA TTT AGT ATT GGA GGT TCT 2761 Tyr Glu Asn Asp Asp Val Glu GlnPhe Gln Phe Ser Ile Gly Gly Ser 785 790 795 TTC TAATAAATTG AACTTTTTTCTTCATCAGAA CTCAAAAACA ACGTTCTCTG 2814 Phe CCTAATTTAA TTGGGCAGAGAAAATATTAA ACCCATCATT TAATTAAGGA TATTTATCAA 2874 ATGAAAAACA TCGCAAAAGTAACCGCACTT GCTTTAGGTA TTGCACTTGC TTCAGGCTAT 2934 GCTTCCGCTG AAGAAAAAATTGCTTTCATT AATGCACTTA TATTTTTCAA 2984 797 amino acids amino acid linearprotein not provided 4 Met Lys Lys Leu Leu Ile Ala Ser Leu Leu Phe GlyThr Thr Thr Thr 1 5 10 15 Val Phe Ala Ala Pro Phe Val Ala Lys Asp IleArg Val Asp Gly Val 20 25 30 Gln Gly Asp Leu Glu Gln Gln Ile Arg Ala SerLeu Pro Val Arg Ala 35 40 45 Gly Gln Arg Val Thr Asp Asn Asp Val Ala AsnIle Val Arg Ser Leu 50 55 60 Phe Val Ser Gly Arg Phe Asp Asp Val Lys AlaHis Gln Glu Gly Asp 65 70 75 80 Val Leu Val Val Ser Val Val Ala Lys SerIle Ile Ser Asp Val Lys 85 90 95 Ile Lys Gly Asn Ser Val Ile Pro Thr GluAla Leu Lys Gln Asn Leu 100 105 110 Asp Ala Asn Gly Phe Lys Val Gly AspVal Leu Ile Arg Glu Lys Leu 115 120 125 Asn Glu Phe Ala Lys Ser Val LysGlu His Tyr Ala Ser Val Gly Arg 130 135 140 Tyr Asn Ala Thr Val Glu ProIle Val Asn Thr Leu Pro Asn Asn Arg 145 150 155 160 Ala Glu Ile Leu IleGln Ile Asn Glu Asp Asp Lys Ala Lys Leu Ala 165 170 175 Ser Leu Thr PheLys Gly Asn Glu Ser Val Ser Ser Ser Thr Leu Gln 180 185 190 Glu Gln MetGlu Leu Gln Pro Asp Ser Trp Trp Lys Leu Trp Gly Asn 195 200 205 Lys PheGlu Gly Ala Gln Phe Glu Lys Asp Leu Gln Ser Ile Arg Asp 210 215 220 TyrTyr Leu Asn Asn Gly Tyr Ala Lys Ala Gln Ile Thr Lys Thr Asp 225 230 235240 Val Gln Leu Asn Asp Glu Lys Thr Lys Val Asn Val Thr Ile Asp Val 245250 255 Asn Glu Gly Leu Gln Tyr Asp Leu Arg Ser Ala Arg Ile Ile Gly Asn260 265 270 Leu Gly Gly Met Ser Ala Glu Leu Glu Pro Leu Leu Ser Ala LeuHis 275 280 285 Leu Asn Asp Thr Phe Arg Arg Ser Asp Ile Ala Asp Val GluAsn Ala 290 295 300 Ile Lys Ala Lys Leu Gly Glu Arg Gly Tyr Gly Ser AlaThr Val Asn 305 310 315 320 Ser Val Pro Asp Phe Asp Asp Ala Asn Lys ThrLeu Ala Ile Thr Leu 325 330 335 Val Val Asp Ala Gly Arg Arg Leu Thr ValArg Gln Leu Arg Phe Glu 340 345 350 Gly Asn Thr Val Ser Ala Asp Ser ThrLeu Arg Gln Glu Met Arg Gln 355 360 365 Gln Glu Gly Thr Trp Tyr Asn SerGln Leu Val Glu Leu Gly Lys Ile 370 375 380 Arg Leu Asp Arg Thr Gly PhePhe Glu Thr Val Glu Asn Arg Ile Asp 385 390 395 400 Pro Ile Asn Gly SerAsn Asp Glu Val Asp Val Val Tyr Lys Val Lys 405 410 415 Glu Arg Asn ThrGly Ser Ile Asn Phe Gly Ile Gly Tyr Gly Thr Glu 420 425 430 Ser Gly IleSer Tyr Gln Ala Ser Val Lys Gln Asp Asn Phe Leu Gly 435 440 445 Thr GlyAla Ala Val Ser Ile Ala Gly Thr Lys Asn Asp Tyr Gly Thr 450 455 460 SerVal Asn Leu Gly Tyr Thr Glu Pro Tyr Phe Thr Lys Asp Gly Val 465 470 475480 Ser Leu Gly Gly Asn Val Phe Phe Glu Asn Tyr Asp Asn Ser Lys Ser 485490 495 Asp Thr Ser Ser Asn Tyr Lys Arg Thr Thr Tyr Gly Ser Asn Val Thr500 505 510 Leu Gly Phe Pro Val Asn Glu Asn Asn Ser Tyr Tyr Val Gly LeuGly 515 520 525 His Thr Tyr Asn Lys Ile Ser Asn Phe Ala Leu Glu Tyr AsnArg Asn 530 535 540 Leu Tyr Ile Gln Ser Met Lys Phe Lys Gly Asn Gly IleLys Thr Asn 545 550 555 560 Asp Phe Asp Phe Ser Phe Gly Trp Asn Tyr AsnSer Leu Asn Arg Gly 565 570 575 Tyr Phe Pro Thr Lys Gly Val Lys Ala SerLeu Gly Gly Arg Val Thr 580 585 590 Ile Pro Gly Ser Asp Asn Lys Tyr TyrLys Leu Ser Ala Asp Val Gln 595 600 605 Gly Phe Tyr Pro Leu Asp Arg AspHis Leu Trp Val Val Ser Ala Lys 610 615 620 Ala Ser Ala Gly Tyr Ala AsnGly Phe Gly Asn Lys Arg Leu Pro Phe 625 630 635 640 Tyr Gln Thr Tyr ThrAla Gly Gly Ile Gly Ser Leu Arg Gly Phe Ala 645 650 655 Tyr Gly Ser IleGly Pro Asn Ala Ile Tyr Ala Glu Tyr Gly Asn Gly 660 665 670 Ser Gly ThrGly Thr Phe Lys Lys Ile Ser Ser Asp Val Ile Gly Gly 675 680 685 Asn AlaIle Ala Thr Ala Ser Ala Glu Leu Ile Val Pro Thr Pro Phe 690 695 700 ValSer Asp Lys Ser Gln Asn Thr Val Arg Thr Ser Leu Phe Val Asp 705 710 715720 Ala Ala Ser Val Trp Asn Thr Lys Trp Lys Ser Asp Lys Asn Gly Leu 725730 735 Glu Ser Asp Val Leu Lys Arg Leu Pro Asp Tyr Gly Lys Ser Ser Arg740 745 750 Ile Arg Ala Ser Thr Gly Val Gly Phe Gln Trp Gln Ser Pro IleGly 755 760 765 Pro Leu Val Phe Ser Tyr Ala Lys Pro Ile Lys Lys Tyr GluAsn Asp 770 775 780 Asp Val Glu Gln Phe Gln Phe Ser Ile Gly Gly Ser Phe785 790 795 2950 base pairs nucleic acid single linear DNA (genomic) notprovided CDS 334..2724 5 AATCACTTAC TGGCGATTTG TCATTAAATA ATTTAAGTGGGCCAATTTCT ATTGCAAAAG 60 GTGCTGGCAC ATCAGCAAAT ATTGGATTGG TGTATTTTTTAAGTTTTATG GCACTGATTA 120 GTGTAAATTT AGGGATTATG AATTTATTTC CATTACCAGTATTAGATGGC GGTCATTTAG 180 TTTTTTTAAC AATGGAAGCT GTTAAAGGAA AACCTGTTTCTGAGCGGGTG CAAAGCATCT 240 GTTATCGAAT TGGCGCAGCA CTGTTATTAA GCTTAACGGTGTTTGCATTA TTTAATGATT 300 TTTTACGTCT ATAATTTATA TAGGATACAA TCG ATG AAAAAA CTT CTA ATC GCA 354 Met Lys Lys Leu Leu Ile Ala 1 5 AGT TTA TTA TTCGGT ACG ACA ACG ACT GTG TTT GCC GCA CCT TTT GTG 402 Ser Leu Leu Phe GlyThr Thr Thr Thr Val Phe Ala Ala Pro Phe Val 10 15 20 GCA AAA GAT ATT CGTGTG GAT GGT GTT CAA GGT GAC TTA GAA CAA CAA 450 Ala Lys Asp Ile Arg ValAsp Gly Val Gln Gly Asp Leu Glu Gln Gln 25 30 35 ATC CGA GCA AGT TTA CCTGTT CGT GCC GGT CAG CGT GTG ACT GAC AAT 498 Ile Arg Ala Ser Leu Pro ValArg Ala Gly Gln Arg Val Thr Asp Asn 40 45 50 55 GAT GTG GCT AAT ATT GTCCGC TCT TTA TTC GTA AGT GGT CGA TTC GAT 546 Asp Val Ala Asn Ile Val ArgSer Leu Phe Val Ser Gly Arg Phe Asp 60 65 70 GAT GTG AAA GCG CAT CAA GAAGGC GAT GTG CTT GTT GTT AGC GTT GTG 594 Asp Val Lys Ala His Gln Glu GlyAsp Val Leu Val Val Ser Val Val 75 80 85 GCT AAA TCG ATC ATT TCA GAT GTTAAA ATC AAA GGT AAC TCT GTT ATT 642 Ala Lys Ser Ile Ile Ser Asp Val LysIle Lys Gly Asn Ser Val Ile 90 95 100 CCC ACT GAA GCA CTT AAA CAA AACTTA GAT GCT AAC GGG TTT AAA GTT 690 Pro Thr Glu Ala Leu Lys Gln Asn LeuAsp Ala Asn Gly Phe Lys Val 105 110 115 GGC GAT GTT TTA ATT CGA GAA AAATTA AAT GAA TTT GCC AAA AGT GTA 738 Gly Asp Val Leu Ile Arg Glu Lys LeuAsn Glu Phe Ala Lys Ser Val 120 125 130 135 AAA GAG CAC TAT GCA AGT GTAGGT CGC TAT AAC GCA ACA GTT GAA CCT 786 Lys Glu His Tyr Ala Ser Val GlyArg Tyr Asn Ala Thr Val Glu Pro 140 145 150 ATT GTC AAT ACG CTA CCA AATAAT CGC GCT GAA ATT TTA ATT CAA ATC 834 Ile Val Asn Thr Leu Pro Asn AsnArg Ala Glu Ile Leu Ile Gln Ile 155 160 165 AAT GAA GAT GAT AAA GCA AAATTG GCA TCA TTA ACT TTC AAG GGG AAC 882 Asn Glu Asp Asp Lys Ala Lys LeuAla Ser Leu Thr Phe Lys Gly Asn 170 175 180 GAA TCT GTT AGT AGC AGT ACATTA CAA GAA CAA ATG GAA TTA CAA CCT 930 Glu Ser Val Ser Ser Ser Thr LeuGln Glu Gln Met Glu Leu Gln Pro 185 190 195 GAT TCT TGG TGG AAA TTA TGGGGA AAT AAA TTT GAA GGT GCG CAA TTC 978 Asp Ser Trp Trp Lys Leu Trp GlyAsn Lys Phe Glu Gly Ala Gln Phe 200 205 210 215 GAG AAA GAT TTG CAG TCAATT CGT GAT TAT TAT TTA AAT AAT GGC TAT 1026 Glu Lys Asp Leu Gln Ser IleArg Asp Tyr Tyr Leu Asn Asn Gly Tyr 220 225 230 GCC AAA GCA CAA ATT ACTAAA ACG GAT GTT CAG CTA AAT GAT GAA AAA 1074 Ala Lys Ala Gln Ile Thr LysThr Asp Val Gln Leu Asn Asp Glu Lys 235 240 245 ACA AAA GTT AAT GTA ACCATT GAT GTA AAT GAA GGT TTA CAG TAT GAC 1122 Thr Lys Val Asn Val Thr IleAsp Val Asn Glu Gly Leu Gln Tyr Asp 250 255 260 CTT CGT AGT GCA CGC ATTATA GGT AAT CTG GGA GGT ATG TCT GCC GAG 1170 Leu Arg Ser Ala Arg Ile IleGly Asn Leu Gly Gly Met Ser Ala Glu 265 270 275 CTT GAA CCT TTA CTT TCAGCA TTA CAT TTA AAT GAT ACT TTC CGC CGT 1218 Leu Glu Pro Leu Leu Ser AlaLeu His Leu Asn Asp Thr Phe Arg Arg 280 285 290 295 AGT GAT ATT GCA GATGTA GAA AAT GCA ATT AAA GCA AAA CTT GGA GAA 1266 Ser Asp Ile Ala Asp ValGlu Asn Ala Ile Lys Ala Lys Leu Gly Glu 300 305 310 CGC GGT TAC GGT AGCGCA ACG GTA AAT TCA GTA CCT GAT TTT GAT GAT 1314 Arg Gly Tyr Gly Ser AlaThr Val Asn Ser Val Pro Asp Phe Asp Asp 315 320 325 GCA AAT AAA ACA TTAGCG ATA ACC CTT GTT GTT GAT GCT GGA CGA CGT 1362 Ala Asn Lys Thr Leu AlaIle Thr Leu Val Val Asp Ala Gly Arg Arg 330 335 340 TTA ACT GTT CGC CAACTT CGC TTT GAA GGA AAT ACC GTT TCT GCT GAT 1410 Leu Thr Val Arg Gln LeuArg Phe Glu Gly Asn Thr Val Ser Ala Asp 345 350 355 AGC ACT TTA CGT CAGGAA ATG CGC CAA CAA GAA GGA ACT TGG TAT AAT 1458 Ser Thr Leu Arg Gln GluMet Arg Gln Gln Glu Gly Thr Trp Tyr Asn 360 365 370 375 TCA CAA TTA GTTGAG TTA GGA AAA ATT CGC TTA GAT CGT ACA GGT TTC 1506 Ser Gln Leu Val GluLeu Gly Lys Ile Arg Leu Asp Arg Thr Gly Phe 380 385 390 TTC GAA ACA GTCGAA AAC CGA ATT GAT CCT ATC AAT GGT AGT AAT GAT 1554 Phe Glu Thr Val GluAsn Arg Ile Asp Pro Ile Asn Gly Ser Asn Asp 395 400 405 GAA GTG GAT GTCGTA TAT AAA GTC AAA GAA CGT AAC ACG GGT AGT ATC 1602 Glu Val Asp Val ValTyr Lys Val Lys Glu Arg Asn Thr Gly Ser Ile 410 415 420 AAC TTT GGT ATTGGT TAC GGT ACA GAG AGT GGT ATT AGT TAT CAA GCA 1650 Asn Phe Gly Ile GlyTyr Gly Thr Glu Ser Gly Ile Ser Tyr Gln Ala 425 430 435 AGT GTT AAA CAAGAT AAT TTC TTG GGA ACA GGG GCG GCA GTA AGT ATA 1698 Ser Val Lys Gln AspAsn Phe Leu Gly Thr Gly Ala Ala Val Ser Ile 440 445 450 455 GCT GGT ACGAAA AAT GAT TAT GGT ACG AGT GTC AAT TTG GGT TAT ACC 1746 Ala Gly Thr LysAsn Asp Tyr Gly Thr Ser Val Asn Leu Gly Tyr Thr 460 465 470 GAG CCC TATTTT ACT AAA GAT GGT GTA AGT CTT GGT GGA AAT GTT TTC 1794 Glu Pro Tyr PheThr Lys Asp Gly Val Ser Leu Gly Gly Asn Val Phe 475 480 485 TTT GAA AACTAC GAT AAC TCT AAA AGT GAT ACA TCC TCT AAC TAT AAG 1842 Phe Glu Asn TyrAsp Asn Ser Lys Ser Asp Thr Ser Ser Asn Tyr Lys 490 495 500 CGT ACG ACTTAC GGA AGT AAT GTT ACT TTA GGT TTC CCT GTA AAT GAA 1890 Arg Thr Thr TyrGly Ser Asn Val Thr Leu Gly Phe Pro Val Asn Glu 505 510 515 AAT AAC TCCTAT TAT GTA GGA TTA GGT CAT ACC TAT AAT AAA ATT AGT 1938 Asn Asn Ser TyrTyr Val Gly Leu Gly His Thr Tyr Asn Lys Ile Ser 520 525 530 535 AAC TTTGCT CTA GAA TAT AAC CGT AAT TTA TAT ATT CAA TCA ATG AAA 1986 Asn Phe AlaLeu Glu Tyr Asn Arg Asn Leu Tyr Ile Gln Ser Met Lys 540 545 550 TTT AAAGGT AAT GGC ATT AAA ACA AAT GAC TTT GAT TTT TCT TTT GGT 2034 Phe Lys GlyAsn Gly Ile Lys Thr Asn Asp Phe Asp Phe Ser Phe Gly 555 560 565 TGG AACTAT AAC AGC CTT AAT AGA GGC TAT TTC CCA ACT AAA GGG GTT 2082 Trp Asn TyrAsn Ser Leu Asn Arg Gly Tyr Phe Pro Thr Lys Gly Val 570 575 580 AAA GCAAGT CTT GGT GGA CGA GTT ACT ATT CCA GGT TCT GAT AAC AAA 2130 Lys Ala SerLeu Gly Gly Arg Val Thr Ile Pro Gly Ser Asp Asn Lys 585 590 595 TAC TACAAA CTA AGT GCA GAT GTA CAG GGT TTC TAC CCA TTA GAC AGA 2178 Tyr Tyr LysLeu Ser Ala Asp Val Gln Gly Phe Tyr Pro Leu Asp Arg 600 605 610 615 GATCAC CTC TGG GTT GTA TCT GCA AAA GCA TCT GCA GGA TAT GCA AAT 2226 Asp HisLeu Trp Val Val Ser Ala Lys Ala Ser Ala Gly Tyr Ala Asn 620 625 630 GGTTTT GGA AAC AAG CGT TTA CCG TTC TAT CAA ACT TAT ACA GCG GGT 2274 Gly PheGly Asn Lys Arg Leu Pro Phe Tyr Gln Thr Tyr Thr Ala Gly 635 640 645 GGCATC GGT TCA TTA CGT GGT TTT GCT TAT GGT AGT ATT GGA CCT AAC 2322 Gly IleGly Ser Leu Arg Gly Phe Ala Tyr Gly Ser Ile Gly Pro Asn 650 655 660 GCAATT TAT GCC GAA TAT GGT AAT GGT AGT GGT ACT GGT ACT TTT AAG 2370 Ala IleTyr Ala Glu Tyr Gly Asn Gly Ser Gly Thr Gly Thr Phe Lys 665 670 675 AAGATA AGT TCT GAT GTG ATT GGT GGT AAT GCA ATC GCT ACA GCT AGC 2418 Lys IleSer Ser Asp Val Ile Gly Gly Asn Ala Ile Ala Thr Ala Ser 680 685 690 695GCA GAG TTA ATT GTG CCA ACT CCA TTT GTG AGC GAT AAG AGC CAA AAT 2466 AlaGlu Leu Ile Val Pro Thr Pro Phe Val Ser Asp Lys Ser Gln Asn 700 705 710ACG GTC CGA ACC TCC TTA TTT GTT GAT GCG GCA AGT GTT TGG AAT ACT 2514 ThrVal Arg Thr Ser Leu Phe Val Asp Ala Ala Ser Val Trp Asn Thr 715 720 725AAA TGG AAA TCA GAT AAA AAT GGA TTA GAG AGC GAT GTA TTA AAA AGA 2562 LysTrp Lys Ser Asp Lys Asn Gly Leu Glu Ser Asp Val Leu Lys Arg 730 735 740TTG CCT GAT TAT GGC AAA TCA AGC CGT ATT CGC GCC TCT ACA GGT GTC 2610 LeuPro Asp Tyr Gly Lys Ser Ser Arg Ile Arg Ala Ser Thr Gly Val 745 750 755GGA TTC CAA TGG CAA TCT CCT ATT GGG CCA TTG GTA TTC TCT TAT GCC 2658 GlyPhe Gln Trp Gln Ser Pro Ile Gly Pro Leu Val Phe Ser Tyr Ala 760 765 770775 AAA CCA ATT AAA AAA TAT GAA AAT GAT GAT GTC GAA CAG TTC CAA TTT 2706Lys Pro Ile Lys Lys Tyr Glu Asn Asp Asp Val Glu Gln Phe Gln Phe 780 785790 AGT ATT GGA GGT TCT TTC TAATAAATTG AACTTTTTTC TTCATCAGAA 2754 SerIle Gly Gly Ser Phe 795 CTCAAAAACA ACGTTCTCTG CCTAATTTAA TTGGGCAGAGAAAATATTAA ACCCATCATT 2814 TAATTAAGGA TATTTATCAA ATGAAAAACA TCGCAAAAGTAACCGCACTT GCTTTAGGTA 2874 TTGCACTTGC TTCAGGCTAT GCTTCCGCTG AAGAAAAAATTGCTTTCATT AATGCGGGTT 2934 ATATTTCAAG GCAAGG 2950 797 amino acids aminoacid linear protein not provided 6 Met Lys Lys Leu Leu Ile Ala Ser LeuLeu Phe Gly Thr Thr Thr Thr 1 5 10 15 Val Phe Ala Ala Pro Phe Val AlaLys Asp Ile Arg Val Asp Gly Val 20 25 30 Gln Gly Asp Leu Glu Gln Gln IleArg Ala Ser Leu Pro Val Arg Ala 35 40 45 Gly Gln Arg Val Thr Asp Asn AspVal Ala Asn Ile Val Arg Ser Leu 50 55 60 Phe Val Ser Gly Arg Phe Asp AspVal Lys Ala His Gln Glu Gly Asp 65 70 75 80 Val Leu Val Val Ser Val ValAla Lys Ser Ile Ile Ser Asp Val Lys 85 90 95 Ile Lys Gly Asn Ser Val IlePro Thr Glu Ala Leu Lys Gln Asn Leu 100 105 110 Asp Ala Asn Gly Phe LysVal Gly Asp Val Leu Ile Arg Glu Lys Leu 115 120 125 Asn Glu Phe Ala LysSer Val Lys Glu His Tyr Ala Ser Val Gly Arg 130 135 140 Tyr Asn Ala ThrVal Glu Pro Ile Val Asn Thr Leu Pro Asn Asn Arg 145 150 155 160 Ala GluIle Leu Ile Gln Ile Asn Glu Asp Asp Lys Ala Lys Leu Ala 165 170 175 SerLeu Thr Phe Lys Gly Asn Glu Ser Val Ser Ser Ser Thr Leu Gln 180 185 190Glu Gln Met Glu Leu Gln Pro Asp Ser Trp Trp Lys Leu Trp Gly Asn 195 200205 Lys Phe Glu Gly Ala Gln Phe Glu Lys Asp Leu Gln Ser Ile Arg Asp 210215 220 Tyr Tyr Leu Asn Asn Gly Tyr Ala Lys Ala Gln Ile Thr Lys Thr Asp225 230 235 240 Val Gln Leu Asn Asp Glu Lys Thr Lys Val Asn Val Thr IleAsp Val 245 250 255 Asn Glu Gly Leu Gln Tyr Asp Leu Arg Ser Ala Arg IleIle Gly Asn 260 265 270 Leu Gly Gly Met Ser Ala Glu Leu Glu Pro Leu LeuSer Ala Leu His 275 280 285 Leu Asn Asp Thr Phe Arg Arg Ser Asp Ile AlaAsp Val Glu Asn Ala 290 295 300 Ile Lys Ala Lys Leu Gly Glu Arg Gly TyrGly Ser Ala Thr Val Asn 305 310 315 320 Ser Val Pro Asp Phe Asp Asp AlaAsn Lys Thr Leu Ala Ile Thr Leu 325 330 335 Val Val Asp Ala Gly Arg ArgLeu Thr Val Arg Gln Leu Arg Phe Glu 340 345 350 Gly Asn Thr Val Ser AlaAsp Ser Thr Leu Arg Gln Glu Met Arg Gln 355 360 365 Gln Glu Gly Thr TrpTyr Asn Ser Gln Leu Val Glu Leu Gly Lys Ile 370 375 380 Arg Leu Asp ArgThr Gly Phe Phe Glu Thr Val Glu Asn Arg Ile Asp 385 390 395 400 Pro IleAsn Gly Ser Asn Asp Glu Val Asp Val Val Tyr Lys Val Lys 405 410 415 GluArg Asn Thr Gly Ser Ile Asn Phe Gly Ile Gly Tyr Gly Thr Glu 420 425 430Ser Gly Ile Ser Tyr Gln Ala Ser Val Lys Gln Asp Asn Phe Leu Gly 435 440445 Thr Gly Ala Ala Val Ser Ile Ala Gly Thr Lys Asn Asp Tyr Gly Thr 450455 460 Ser Val Asn Leu Gly Tyr Thr Glu Pro Tyr Phe Thr Lys Asp Gly Val465 470 475 480 Ser Leu Gly Gly Asn Val Phe Phe Glu Asn Tyr Asp Asn SerLys Ser 485 490 495 Asp Thr Ser Ser Asn Tyr Lys Arg Thr Thr Tyr Gly SerAsn Val Thr 500 505 510 Leu Gly Phe Pro Val Asn Glu Asn Asn Ser Tyr TyrVal Gly Leu Gly 515 520 525 His Thr Tyr Asn Lys Ile Ser Asn Phe Ala LeuGlu Tyr Asn Arg Asn 530 535 540 Leu Tyr Ile Gln Ser Met Lys Phe Lys GlyAsn Gly Ile Lys Thr Asn 545 550 555 560 Asp Phe Asp Phe Ser Phe Gly TrpAsn Tyr Asn Ser Leu Asn Arg Gly 565 570 575 Tyr Phe Pro Thr Lys Gly ValLys Ala Ser Leu Gly Gly Arg Val Thr 580 585 590 Ile Pro Gly Ser Asp AsnLys Tyr Tyr Lys Leu Ser Ala Asp Val Gln 595 600 605 Gly Phe Tyr Pro LeuAsp Arg Asp His Leu Trp Val Val Ser Ala Lys 610 615 620 Ala Ser Ala GlyTyr Ala Asn Gly Phe Gly Asn Lys Arg Leu Pro Phe 625 630 635 640 Tyr GlnThr Tyr Thr Ala Gly Gly Ile Gly Ser Leu Arg Gly Phe Ala 645 650 655 TyrGly Ser Ile Gly Pro Asn Ala Ile Tyr Ala Glu Tyr Gly Asn Gly 660 665 670Ser Gly Thr Gly Thr Phe Lys Lys Ile Ser Ser Asp Val Ile Gly Gly 675 680685 Asn Ala Ile Ala Thr Ala Ser Ala Glu Leu Ile Val Pro Thr Pro Phe 690695 700 Val Ser Asp Lys Ser Gln Asn Thr Val Arg Thr Ser Leu Phe Val Asp705 710 715 720 Ala Ala Ser Val Trp Asn Thr Lys Trp Lys Ser Asp Lys AsnGly Leu 725 730 735 Glu Ser Asp Val Leu Lys Arg Leu Pro Asp Tyr Gly LysSer Ser Arg 740 745 750 Ile Arg Ala Ser Thr Gly Val Gly Phe Gln Trp GlnSer Pro Ile Gly 755 760 765 Pro Leu Val Phe Ser Tyr Ala Lys Pro Ile LysLys Tyr Glu Asn Asp 770 775 780 Asp Val Glu Gln Phe Gln Phe Ser Ile GlyGly Ser Phe 785 790 795 2974 base pairs nucleic acid single linear DNA(genomic) not provided CDS 386..2761 7 GGCATTGAAA AAACAGGACA GCTTTCCCTTTTAACCTTGA AAATATTAGG GAAATTACTT 60 ACTGGCGATT TGTCATTAAA TAATTTAAGTGGGCCAATTT CTATTGCAAA AGGTGCTGGC 120 GCATCAGCAA ATATTGGATT GGTGTATTTTTTAAGTTTTA TGGCATTGAT TAGTGTAAAT 180 TTAGGGATTA TGAATTTATT TCCATTACCAGTATTAGATG GCGGTCATTT AGTTTTTTTA 240 ACAATGGAAG CTGTTAAAGG AAAACCTGTTTCTGAGCGGG TGCAAAGCAT CTGTTATCGA 300 ATTGGCGCAG CACTGTTATT AAGCTTAACGGTGTTTGCAT TATTTAATGA TTTTTTACGT 360 CTATAATTTA TATAGGATAC AATCG ATG AAAAAA CTT CTA ATC GCA AGT TTA 412 Met Lys Lys Leu Leu Ile Ala Ser Leu 1 5TTA TTC GGT ACG ACA ACG ACT GTG TTT GCC GCA CCT TTT GTG GCA AAA 460 LeuPhe Gly Thr Thr Thr Thr Val Phe Ala Ala Pro Phe Val Ala Lys 10 15 20 25GAT ATT CGT GTG GAT GGT GTT CAA GGT GAC TTA GAA CAA CAA ATC CGA 508 AspIle Arg Val Asp Gly Val Gln Gly Asp Leu Glu Gln Gln Ile Arg 30 35 40 GCAAGT TTA CCT GTT CGT GCC GGT CAG CGT GTG ACT GAC AAT GAT GTG 556 Ala SerLeu Pro Val Arg Ala Gly Gln Arg Val Thr Asp Asn Asp Val 45 50 55 GCT AATATT GTC CGC TCT TTA TTC GTA AGT GGT CGA TTC GAT GAT GTG 604 Ala Asn IleVal Arg Ser Leu Phe Val Ser Gly Arg Phe Asp Asp Val 60 65 70 AAA GCG CATCAA GAA GGC GAT GTG CTT GTT GTT AGC GTT GTG GCT AAA 652 Lys Ala His GlnGlu Gly Asp Val Leu Val Val Ser Val Val Ala Lys 75 80 85 TCG ATC ATT TCAGAT GTT AAA ATC AAA GGT AAC TCT ATT ATT CCA CCT 700 Ser Ile Ile Ser AspVal Lys Ile Lys Gly Asn Ser Ile Ile Pro Pro 90 95 100 105 GAA GCA CTAAAA CAA AAC TTA GAT GCT AAC GGG TTT AAA GTT GGC GAT 748 Glu Ala Leu LysGln Asn Leu Asp Ala Asn Gly Phe Lys Val Gly Asp 110 115 120 ATT TTA ATTCGA GAA AAA TTA AAT GAA TTT GCC CAA AGT GTA AAA GAG 796 Ile Leu Ile ArgGlu Lys Leu Asn Glu Phe Ala Gln Ser Val Lys Glu 125 130 135 CAC TAT GCAAGT GTA GGT CGC TAT AAC GCA ACC GTT GAA CCT ATT GTC 844 His Tyr Ala SerVal Gly Arg Tyr Asn Ala Thr Val Glu Pro Ile Val 140 145 150 AAT ACG CTACCA AAT AAT CGC GCT GAA ATT TTA ATT CAA ATC AAT GAA 892 Asn Thr Leu ProAsn Asn Arg Ala Glu Ile Leu Ile Gln Ile Asn Glu 155 160 165 GAT GAT AAAGCC AAA TTG GCA TCA TTA ACT TTC AAG GGG AAC GAA TCT 940 Asp Asp Lys AlaLys Leu Ala Ser Leu Thr Phe Lys Gly Asn Glu Ser 170 175 180 185 GTT AGTAGC AGT ACA TTA CAA GAA CAA ATG GAA TTA CAA CCT GAT TCT 988 Val Ser SerSer Thr Leu Gln Glu Gln Met Glu Leu Gln Pro Asp Ser 190 195 200 TGG TGGAAA TTA TGG GGA AAT AAA TTT GAA GGT GCG CAA TTC GAG AAA 1036 Trp Trp LysLeu Trp Gly Asn Lys Phe Glu Gly Ala Gln Phe Glu Lys 205 210 215 GAT TTGCAG GCA ATT CGT GAT TAT TAT TTA AAT AAT GGC TAT GCC AAA 1084 Asp Leu GlnAla Ile Arg Asp Tyr Tyr Leu Asn Asn Gly Tyr Ala Lys 220 225 230 GCA CAAATC ACT AAA GCG GAT GTT CAG CTA AAT GAT GAA AAA ACA AAA 1132 Ala Gln IleThr Lys Ala Asp Val Gln Leu Asn Asp Glu Lys Thr Lys 235 240 245 GTT AATGTA ACC ATT GAT GTA AAT GAA GGT TTA CAG TAT GAC CTT CGT 1180 Val Asn ValThr Ile Asp Val Asn Glu Gly Leu Gln Tyr Asp Leu Arg 250 255 260 265 AGTGCA CGC ATT ATA GGT AAT CTG GGA GGT ATG TCT GCC GAG CTT GAA 1228 Ser AlaArg Ile Ile Gly Asn Leu Gly Gly Met Ser Ala Glu Leu Glu 270 275 280 CCTTTA CTT TCA GCA TTA CAT TTA AAT GAT ACT TTC CGC CGT AGT GAT 1276 Pro LeuLeu Ser Ala Leu His Leu Asn Asp Thr Phe Arg Arg Ser Asp 285 290 295 ATTGCA GAT GTA GAA AAT GCA ATT AAA GCA AAA CTT GGG GAA CGA GGT 1324 Ile AlaAsp Val Glu Asn Ala Ile Lys Ala Lys Leu Gly Glu Arg Gly 300 305 310 TACGGT AAC ACA ACA GTA AAT TCT GTA CCT GAT TTT GAC GAT GCA AAT 1372 Tyr GlyAsn Thr Thr Val Asn Ser Val Pro Asp Phe Asp Asp Ala Asn 315 320 325 AAAACA TTA GCG ATA ACC TTT GTT GTT GAT GCT GGA CGA CGT TTA ACT 1420 Lys ThrLeu Ala Ile Thr Phe Val Val Asp Ala Gly Arg Arg Leu Thr 330 335 340 345GTT CAC CAA CTT CGC TTT GAA GGA AAT ACC GTT TCT GCT GAT AGT ACT 1468 ValHis Gln Leu Arg Phe Glu Gly Asn Thr Val Ser Ala Asp Ser Thr 350 355 360TTA CGT CAG GAA ATG CGC CAA CAA GAA GGA ACT TGG TAT AAT TCA CAA 1516 LeuArg Gln Glu Met Arg Gln Gln Glu Gly Thr Trp Tyr Asn Ser Gln 365 370 375TTA GTT GAG TTA GGA AAA ATT CGC TTA GAT CGT ACA GGT TTC TTC GAA 1564 LeuVal Glu Leu Gly Lys Ile Arg Leu Asp Arg Thr Gly Phe Phe Glu 380 385 390ACA GTT GAA AAC CGA ATT GAT CCT ATC AAT GGT AGC AAT GAT GAA GTG 1612 ThrVal Glu Asn Arg Ile Asp Pro Ile Asn Gly Ser Asn Asp Glu Val 395 400 405GAT GTC GTA TAT AAA GTC AAA GAA CGT AAC ACG GGT AGT ATC AAC TTT 1660 AspVal Val Tyr Lys Val Lys Glu Arg Asn Thr Gly Ser Ile Asn Phe 410 415 420425 GGT ATT GGT TAC GGT ACA GAG AGT GGT ATT AGT TAT CAA GCA AGT GTC 1708Gly Ile Gly Tyr Gly Thr Glu Ser Gly Ile Ser Tyr Gln Ala Ser Val 430 435440 AAA CAA GAT AAT TTC TTG GGA ACA GGG GCG GCA GTA AGT ATA GCT GGT 1756Lys Gln Asp Asn Phe Leu Gly Thr Gly Ala Ala Val Ser Ile Ala Gly 445 450455 ACG AAA AAT GAT TAT GGT ACG AGT GTC AAT TTG GGT TAT ACC GAG CCC 1804Thr Lys Asn Asp Tyr Gly Thr Ser Val Asn Leu Gly Tyr Thr Glu Pro 460 465470 TAT TTT ACT AAA GAT GGT GTA AGT CTT GGT GGA AAT GTT TTC TTT GAA 1852Tyr Phe Thr Lys Asp Gly Val Ser Leu Gly Gly Asn Val Phe Phe Glu 475 480485 AAC TAC GAT AAC TCT AAA AGT GAT ACA TCC TCT AAC TAT AAG CGT ACG 1900Asn Tyr Asp Asn Ser Lys Ser Asp Thr Ser Ser Asn Tyr Lys Arg Thr 490 495500 505 ACT TAT GGA AGT AAT GTT ACT TTA GGT TTC CCT GTA AAT GAA AAT AAC1948 Thr Tyr Gly Ser Asn Val Thr Leu Gly Phe Pro Val Asn Glu Asn Asn 510515 520 TCC TAT TAT GTA GGA TTA GGC CAT ACC TAT AAT AAA ATT AGT AAC TTT1996 Ser Tyr Tyr Val Gly Leu Gly His Thr Tyr Asn Lys Ile Ser Asn Phe 525530 535 GCT CTA GAA TAT AAC CGT AAT TTA TAT ATT CAA TCA ATG AAA TTT AAA2044 Ala Leu Glu Tyr Asn Arg Asn Leu Tyr Ile Gln Ser Met Lys Phe Lys 540545 550 GGT AAT GGC ATT AAA ACA AAT GAC TTT GAT TTT TCT TTT GGT TGG AAC2092 Gly Asn Gly Ile Lys Thr Asn Asp Phe Asp Phe Ser Phe Gly Trp Asn 555560 565 TAT AAC AGC CTT AAT AGA GGC TAT TTC CCA ACT AAA GGG GTT AAA GCA2140 Tyr Asn Ser Leu Asn Arg Gly Tyr Phe Pro Thr Lys Gly Val Lys Ala 570575 580 585 AGT CTT GGT GGA CGA GTT ACA ATT CCA GGT TCT GAT AAC AAA TACTAC 2188 Ser Leu Gly Gly Arg Val Thr Ile Pro Gly Ser Asp Asn Lys Tyr Tyr590 595 600 AAA CTA AGT GCA GAT GTA CAG GGT TTC TAC CCA TTA GAC AGA GATCAC 2236 Lys Leu Ser Ala Asp Val Gln Gly Phe Tyr Pro Leu Asp Arg Asp His605 610 615 CTC TGG GTT GTA TCT GCA AAA GCA TCT GCA GGA TAT GCA AAT GGTTTT 2284 Leu Trp Val Val Ser Ala Lys Ala Ser Ala Gly Tyr Ala Asn Gly Phe620 625 630 GGA AAC AAG CGT TTA CCG TTC TAT CAA ACT TAT ACA GCG GGT GGCATT 2332 Gly Asn Lys Arg Leu Pro Phe Tyr Gln Thr Tyr Thr Ala Gly Gly Ile635 640 645 GGT TCA TTA CGC GGT TTT GCT TAT GGT AGC ATT GGG CCT AAC GCAATT 2380 Gly Ser Leu Arg Gly Phe Ala Tyr Gly Ser Ile Gly Pro Asn Ala Ile650 655 660 665 TAT CAA GGT CAA AAT AAT AAA TTT AAT AAG ATA AGT TCT GATGTG ATT 2428 Tyr Gln Gly Gln Asn Asn Lys Phe Asn Lys Ile Ser Ser Asp ValIle 670 675 680 GGT GGT AAT GCA ATC GCT ACA GCT AGC GCA GAG TTA ATT GTGCCA ACT 2476 Gly Gly Asn Ala Ile Ala Thr Ala Ser Ala Glu Leu Ile Val ProThr 685 690 695 CCA TTT GTG AGT GAT AAG AGT CAA AAT ACA GTC CGA ACC TCCCTA TTT 2524 Pro Phe Val Ser Asp Lys Ser Gln Asn Thr Val Arg Thr Ser LeuPhe 700 705 710 GTT GAT GCG GCA AGT GTT TGG AAT ACT AAA TGG AAA TCA GATAAA AAT 2572 Val Asp Ala Ala Ser Val Trp Asn Thr Lys Trp Lys Ser Asp LysAsn 715 720 725 GGA TTA GAG AGC AAT GTC TTG AAA GAC TTA CCC GAT TAT GGCAAA TCA 2620 Gly Leu Glu Ser Asn Val Leu Lys Asp Leu Pro Asp Tyr Gly LysSer 730 735 740 745 AGC CGT ACT CGC GCC TCT ACA GGT GTC GGA TTC CAA TGGCAA TCT CCT 2668 Ser Arg Thr Arg Ala Ser Thr Gly Val Gly Phe Gln Trp GlnSer Pro 750 755 760 AGT GGA CCA GTG GTA TTT TCT TAT GCT AAA CCA ATT AAAAAA TAT GAA 2716 Ser Gly Pro Val Val Phe Ser Tyr Ala Lys Pro Ile Lys LysTyr Glu 765 770 775 AAT GAT GAT GTC GAA CAG TTC CAA TTT AGT ATT GGG GGTTCT TTC 2761 Asn Asp Asp Val Glu Gln Phe Gln Phe Ser Ile Gly Gly Ser Phe780 785 790 TAATAAATTG AACTTTTTTC GTCATCAGAA CTCAAAAACA ACGTTCTCTGCCTAATTTAA 2821 TTGGGCAGAG AAAATATTAA AACCATCATT TAATTAAGGA TATTTATCAAATGAAAAACA 2881 TCGCCAAAGT AACCGCACTT GCTTTAGGTA TTGCACTTGC TTCAGGCTATGCTGCAGCTG 2941 AAGAAAAAAT TGCTTTTATT AATGCAGGTT ATA 2974 792 aminoacids amino acid linear protein not provided 8 Met Lys Lys Leu Leu IleAla Ser Leu Leu Phe Gly Thr Thr Thr Thr 1 5 10 15 Val Phe Ala Ala ProPhe Val Ala Lys Asp Ile Arg Val Asp Gly Val 20 25 30 Gln Gly Asp Leu GluGln Gln Ile Arg Ala Ser Leu Pro Val Arg Ala 35 40 45 Gly Gln Arg Val ThrAsp Asn Asp Val Ala Asn Ile Val Arg Ser Leu 50 55 60 Phe Val Ser Gly ArgPhe Asp Asp Val Lys Ala His Gln Glu Gly Asp 65 70 75 80 Val Leu Val ValSer Val Val Ala Lys Ser Ile Ile Ser Asp Val Lys 85 90 95 Ile Lys Gly AsnSer Ile Ile Pro Pro Glu Ala Leu Lys Gln Asn Leu 100 105 110 Asp Ala AsnGly Phe Lys Val Gly Asp Ile Leu Ile Arg Glu Lys Leu 115 120 125 Asn GluPhe Ala Gln Ser Val Lys Glu His Tyr Ala Ser Val Gly Arg 130 135 140 TyrAsn Ala Thr Val Glu Pro Ile Val Asn Thr Leu Pro Asn Asn Arg 145 150 155160 Ala Glu Ile Leu Ile Gln Ile Asn Glu Asp Asp Lys Ala Lys Leu Ala 165170 175 Ser Leu Thr Phe Lys Gly Asn Glu Ser Val Ser Ser Ser Thr Leu Gln180 185 190 Glu Gln Met Glu Leu Gln Pro Asp Ser Trp Trp Lys Leu Trp GlyAsn 195 200 205 Lys Phe Glu Gly Ala Gln Phe Glu Lys Asp Leu Gln Ala IleArg Asp 210 215 220 Tyr Tyr Leu Asn Asn Gly Tyr Ala Lys Ala Gln Ile ThrLys Ala Asp 225 230 235 240 Val Gln Leu Asn Asp Glu Lys Thr Lys Val AsnVal Thr Ile Asp Val 245 250 255 Asn Glu Gly Leu Gln Tyr Asp Leu Arg SerAla Arg Ile Ile Gly Asn 260 265 270 Leu Gly Gly Met Ser Ala Glu Leu GluPro Leu Leu Ser Ala Leu His 275 280 285 Leu Asn Asp Thr Phe Arg Arg SerAsp Ile Ala Asp Val Glu Asn Ala 290 295 300 Ile Lys Ala Lys Leu Gly GluArg Gly Tyr Gly Asn Thr Thr Val Asn 305 310 315 320 Ser Val Pro Asp PheAsp Asp Ala Asn Lys Thr Leu Ala Ile Thr Phe 325 330 335 Val Val Asp AlaGly Arg Arg Leu Thr Val His Gln Leu Arg Phe Glu 340 345 350 Gly Asn ThrVal Ser Ala Asp Ser Thr Leu Arg Gln Glu Met Arg Gln 355 360 365 Gln GluGly Thr Trp Tyr Asn Ser Gln Leu Val Glu Leu Gly Lys Ile 370 375 380 ArgLeu Asp Arg Thr Gly Phe Phe Glu Thr Val Glu Asn Arg Ile Asp 385 390 395400 Pro Ile Asn Gly Ser Asn Asp Glu Val Asp Val Val Tyr Lys Val Lys 405410 415 Glu Arg Asn Thr Gly Ser Ile Asn Phe Gly Ile Gly Tyr Gly Thr Glu420 425 430 Ser Gly Ile Ser Tyr Gln Ala Ser Val Lys Gln Asp Asn Phe LeuGly 435 440 445 Thr Gly Ala Ala Val Ser Ile Ala Gly Thr Lys Asn Asp TyrGly Thr 450 455 460 Ser Val Asn Leu Gly Tyr Thr Glu Pro Tyr Phe Thr LysAsp Gly Val 465 470 475 480 Ser Leu Gly Gly Asn Val Phe Phe Glu Asn TyrAsp Asn Ser Lys Ser 485 490 495 Asp Thr Ser Ser Asn Tyr Lys Arg Thr ThrTyr Gly Ser Asn Val Thr 500 505 510 Leu Gly Phe Pro Val Asn Glu Asn AsnSer Tyr Tyr Val Gly Leu Gly 515 520 525 His Thr Tyr Asn Lys Ile Ser AsnPhe Ala Leu Glu Tyr Asn Arg Asn 530 535 540 Leu Tyr Ile Gln Ser Met LysPhe Lys Gly Asn Gly Ile Lys Thr Asn 545 550 555 560 Asp Phe Asp Phe SerPhe Gly Trp Asn Tyr Asn Ser Leu Asn Arg Gly 565 570 575 Tyr Phe Pro ThrLys Gly Val Lys Ala Ser Leu Gly Gly Arg Val Thr 580 585 590 Ile Pro GlySer Asp Asn Lys Tyr Tyr Lys Leu Ser Ala Asp Val Gln 595 600 605 Gly PheTyr Pro Leu Asp Arg Asp His Leu Trp Val Val Ser Ala Lys 610 615 620 AlaSer Ala Gly Tyr Ala Asn Gly Phe Gly Asn Lys Arg Leu Pro Phe 625 630 635640 Tyr Gln Thr Tyr Thr Ala Gly Gly Ile Gly Ser Leu Arg Gly Phe Ala 645650 655 Tyr Gly Ser Ile Gly Pro Asn Ala Ile Tyr Gln Gly Gln Asn Asn Lys660 665 670 Phe Asn Lys Ile Ser Ser Asp Val Ile Gly Gly Asn Ala Ile AlaThr 675 680 685 Ala Ser Ala Glu Leu Ile Val Pro Thr Pro Phe Val Ser AspLys Ser 690 695 700 Gln Asn Thr Val Arg Thr Ser Leu Phe Val Asp Ala AlaSer Val Trp 705 710 715 720 Asn Thr Lys Trp Lys Ser Asp Lys Asn Gly LeuGlu Ser Asn Val Leu 725 730 735 Lys Asp Leu Pro Asp Tyr Gly Lys Ser SerArg Thr Arg Ala Ser Thr 740 745 750 Gly Val Gly Phe Gln Trp Gln Ser ProSer Gly Pro Val Val Phe Ser 755 760 765 Tyr Ala Lys Pro Ile Lys Lys TyrGlu Asn Asp Asp Val Glu Gln Phe 770 775 780 Gln Phe Ser Ile Gly Gly SerPhe 785 790 2989 base pairs nucleic acid single linear DNA (genomic) notprovided CDS 390..2768 9 AAAAGGCATT GAAAAAACAG GACAACTTTC CCTTTTAACCTTGAAAATAT TAGGGAAATT 60 ACTTACTGGC GATTTGTCAT TAAATAATTT AAGTGGGCCAATTTCTATTG CAAAAGGTGC 120 TGGTGCATCA GCAAATATTG GATTGGTGTA TTTTTTAAGTTTTATGGCAT TGATTAGTGT 180 AAATTTAGGG ATTATGAATT TATTTCCATT ACCAGTATTAGATGGCGGTC ATTTAGTTTT 240 TTTAACAATG GAAGCTGTTA AAGGAAAACC TGTTTCTGAGCGGGTGCAAA GCATCTGTTA 300 TCGAATTGGC GCAGCACTGT TATTAAGCTT AACGGTGTTTGCATTATTTA ATGATTTTTT 360 ACGTCTATAA TTTATATAGG ATACAATCG ATG AAA AAACTT CTA ATC GCA AGT 413 Met Lys Lys Leu Leu Ile Ala Ser 1 5 TTA TTA TTCGGT GCG ACA ACG ACT GTG TTT GCC GCA CCT TTT GTG CCA 461 Leu Leu Phe GlyAla Thr Thr Thr Val Phe Ala Ala Pro Phe Val Pro 10 15 20 AAA GAT ATT CGTGTG GAT GGT GTT CAA GGT GAC TTA GAA CAA CAA ATC 509 Lys Asp Ile Arg ValAsp Gly Val Gln Gly Asp Leu Glu Gln Gln Ile 25 30 35 40 CGA GCA AGT TTACCT GTT CGT GCT GGT CAG CGT GTG ACT GAC AAT GAT 557 Arg Ala Ser Leu ProVal Arg Ala Gly Gln Arg Val Thr Asp Asn Asp 45 50 55 GTG GCT AAT ATT GTCCGC TCT TTA TTC GTA AGT GGT CGA TTC GAT GAT 605 Val Ala Asn Ile Val ArgSer Leu Phe Val Ser Gly Arg Phe Asp Asp 60 65 70 GTG AAA GCG CAT CAA GAAGGC GAT GTG CTT GTT GTT AGC GTT GTG GCT 653 Val Lys Ala His Gln Glu GlyAsp Val Leu Val Val Ser Val Val Ala 75 80 85 AAA TCG ATC ATT TCA GAT GTTAAA ATC AAA GGT AAC TCT GTT ATT CCC 701 Lys Ser Ile Ile Ser Asp Val LysIle Lys Gly Asn Ser Val Ile Pro 90 95 100 ACT GAA GCA CTT AAA CAA AACTTA GAT GCT AAC GGG TTT AAA GTT GGC 749 Thr Glu Ala Leu Lys Gln Asn LeuAsp Ala Asn Gly Phe Lys Val Gly 105 110 115 120 GAT GTT TTA ATT CGA GAAAAA TTA AAT GAA TTT GCC AAA AGT GTA AAA 797 Asp Val Leu Ile Arg Glu LysLeu Asn Glu Phe Ala Lys Ser Val Lys 125 130 135 GAG CAC TAT GCA AGT GTAGGT CGC TAT AAC GCA ACC GTT GAA CCT ATT 845 Glu His Tyr Ala Ser Val GlyArg Tyr Asn Ala Thr Val Glu Pro Ile 140 145 150 GTC AAT ACG CTG CCA AATAAT CGT GCT GAA ATT TTA ATT CAA ATC AAT 893 Val Asn Thr Leu Pro Asn AsnArg Ala Glu Ile Leu Ile Gln Ile Asn 155 160 165 GAA GAT GAT AAA GCA AAATTG GCA TCA TTA ACT TTC AAG GGG AAC GAA 941 Glu Asp Asp Lys Ala Lys LeuAla Ser Leu Thr Phe Lys Gly Asn Glu 170 175 180 TCT GTT AGT AGC AGT ACATTA CAA GAA CAA ATG GAA TTA CAA CCT GAT 989 Ser Val Ser Ser Ser Thr LeuGln Glu Gln Met Glu Leu Gln Pro Asp 185 190 195 200 TCT TGG TGG AAA TTATGG GGA AAT AAA TTT GAA GGT GCG CAA TTC GAG 1037 Ser Trp Trp Lys Leu TrpGly Asn Lys Phe Glu Gly Ala Gln Phe Glu 205 210 215 AAA GAT CTG CAG GCAATT CGT GAT TAT TAT TTA AAT AAT GGC TAT GCC 1085 Lys Asp Leu Gln Ala IleArg Asp Tyr Tyr Leu Asn Asn Gly Tyr Ala 220 225 230 AAA GCA CAA ATC ACTAAA ACG GAT GTT CAG CTA AAT GAT GAA AAA ACA 1133 Lys Ala Gln Ile Thr LysThr Asp Val Gln Leu Asn Asp Glu Lys Thr 235 240 245 AAA GTT AAT GTA ACCATT GAT GTA AAT GAA GGT TTA CAG TAT GAC CTT 1181 Lys Val Asn Val Thr IleAsp Val Asn Glu Gly Leu Gln Tyr Asp Leu 250 255 260 CGT AGT GCA CGC ATTATA GGT AAT CTG GGA GGT ATG TCT GCC GAG CTT 1229 Arg Ser Ala Arg Ile IleGly Asn Leu Gly Gly Met Ser Ala Glu Leu 265 270 275 280 GAA CCT TTA CTTTCA GCA TTA CAT TTA AAT GAT ACT TTC CGC CGT AGT 1277 Glu Pro Leu Leu SerAla Leu His Leu Asn Asp Thr Phe Arg Arg Ser 285 290 295 GAT ATT GCA GATGTA GAA AAT GCA ATT AAA GCA AAA CTT GGG GAA CGA 1325 Asp Ile Ala Asp ValGlu Asn Ala Ile Lys Ala Lys Leu Gly Glu Arg 300 305 310 GGT TAC GGT AACACA ACA GTA AAT TCT GTA CCT GAT TTT GAC GAT GCA 1373 Gly Tyr Gly Asn ThrThr Val Asn Ser Val Pro Asp Phe Asp Asp Ala 315 320 325 AAT AAA ACA TTAGCG ATA ACC TTT GTT GTT GAT GCT GGA CGA CGT TTA 1421 Asn Lys Thr Leu AlaIle Thr Phe Val Val Asp Ala Gly Arg Arg Leu 330 335 340 ACT GTT CGC CAACTT CGC TTT GAA GGA AAT ACC GTT TCT GCT GAT AGT 1469 Thr Val Arg Gln LeuArg Phe Glu Gly Asn Thr Val Ser Ala Asp Ser 345 350 355 360 ACT TTA CGTCAG GAA ATG CGA CAA CAA GAA GGA ACT TGG TAT AAT TCA 1517 Thr Leu Arg GlnGlu Met Arg Gln Gln Glu Gly Thr Trp Tyr Asn Ser 365 370 375 CAA TTA GTTGAG TTA GGA AAA ATT CGC TTA GAT CGT ACA GGT TTC TTC 1565 Gln Leu Val GluLeu Gly Lys Ile Arg Leu Asp Arg Thr Gly Phe Phe 380 385 390 GAA ACA GTTGAA AAC CGA ATT GAT CCT ATC AAT GGT AGC AAT GAT GAA 1613 Glu Thr Val GluAsn Arg Ile Asp Pro Ile Asn Gly Ser Asn Asp Glu 395 400 405 GTG GAT GTCGTA TAT AAA GTC AAA GAA CGT AAC ACG GGT AGT ATC AAC 1661 Val Asp Val ValTyr Lys Val Lys Glu Arg Asn Thr Gly Ser Ile Asn 410 415 420 TTT GGT ATTGGT TAC GGT ACA GAG AGT GGT ATC AGT TAT CAA ACA AGT 1709 Phe Gly Ile GlyTyr Gly Thr Glu Ser Gly Ile Ser Tyr Gln Thr Ser 425 430 435 440 ATT AAACAA GAT AAT TTC TTG GGA ACA GGG GCG GCA GTA AGT ATA GCT 1757 Ile Lys GlnAsp Asn Phe Leu Gly Thr Gly Ala Ala Val Ser Ile Ala 445 450 455 GGT ACGAAA AAT GAT TAT GGT ACG AGT GTC AAT TTG GGT TAT ACC GAA 1805 Gly Thr LysAsn Asp Tyr Gly Thr Ser Val Asn Leu Gly Tyr Thr Glu 460 465 470 CCC TATTTT ACT AAA GAT GGT GTA AGT CTT GGT GGA AAT ATT TTC TTT 1853 Pro Tyr PheThr Lys Asp Gly Val Ser Leu Gly Gly Asn Ile Phe Phe 475 480 485 GAA AACTAC GAT AAC TCT AAA AGT GAT ACA TCC TCT AAC TAT AAG CGT 1901 Glu Asn TyrAsp Asn Ser Lys Ser Asp Thr Ser Ser Asn Tyr Lys Arg 490 495 500 ACG ACTTAT GGA AGT AAT GTT ACT TTA GGT TTC CCT GTA AAT GAA AAT 1949 Thr Thr TyrGly Ser Asn Val Thr Leu Gly Phe Pro Val Asn Glu Asn 505 510 515 520 AACTCC TAT TAT GTA GGA TTA GGC CAT ACC TAT AAT AAA ATT AGT AAC 1997 Asn SerTyr Tyr Val Gly Leu Gly His Thr Tyr Asn Lys Ile Ser Asn 525 530 535 TTTGCT CTA GAA TAT AAC CGT AAT TTA TAT ATT CAA TCA ATG AAA TTT 2045 Phe AlaLeu Glu Tyr Asn Arg Asn Leu Tyr Ile Gln Ser Met Lys Phe 540 545 550 AAAGGT AAT GGC ATT AAA ACA AAT GAC TTT GAT TTT TCT TTT GGT TGG 2093 Lys GlyAsn Gly Ile Lys Thr Asn Asp Phe Asp Phe Ser Phe Gly Trp 555 560 565 AACTAT AAC AGC CTT AAT AGA GGC TAT TTC CCA ACT AAA GGG GTT AAA 2141 Asn TyrAsn Ser Leu Asn Arg Gly Tyr Phe Pro Thr Lys Gly Val Lys 570 575 580 GCAAGT CTT GGT GGA CGA GTT ACT ATT CCA GGT TCT GAT AAC AAA TAC 2189 Ala SerLeu Gly Gly Arg Val Thr Ile Pro Gly Ser Asp Asn Lys Tyr 585 590 595 600TAC AAA CTA AGT GCA GAT GTA CAG GGT TTC TAC CCA TTA GAC AGA GAT 2237 TyrLys Leu Ser Ala Asp Val Gln Gly Phe Tyr Pro Leu Asp Arg Asp 605 610 615CAC CGC TGG GTT GTA TCT GCA AAA GCA TCT GCA GGA TAT GCA AAT GGT 2285 HisArg Trp Val Val Ser Ala Lys Ala Ser Ala Gly Tyr Ala Asn Gly 620 625 630TTT GGA AAC AAG CGT TTA CCG TTC TAT CAA ACT TAT ACA GCG GGT GGC 2333 PheGly Asn Lys Arg Leu Pro Phe Tyr Gln Thr Tyr Thr Ala Gly Gly 635 640 645ATT GGT TCA TTA CGC GGT TTT GCT TAT GGT AGT ATT GGG CCT AAT GCA 2381 IleGly Ser Leu Arg Gly Phe Ala Tyr Gly Ser Ile Gly Pro Asn Ala 650 655 660ATT TAT GCC GAA CAT GGT AAT GGT ACT TTT AAT AAG ATA AGT TCT GAT 2429 IleTyr Ala Glu His Gly Asn Gly Thr Phe Asn Lys Ile Ser Ser Asp 665 670 675680 GTG ATT GGT GGT AAT GCA ATC ACA ACT GCG AGT GCA GAA CTT ATT GTA 2477Val Ile Gly Gly Asn Ala Ile Thr Thr Ala Ser Ala Glu Leu Ile Val 685 690695 CCA ACT CCA TTT GTG AGT GAT AAA AGC CAA AAT ACA GTC CGA ACC TCC 2525Pro Thr Pro Phe Val Ser Asp Lys Ser Gln Asn Thr Val Arg Thr Ser 700 705710 CTA TTT GTT GAT GCG GCA AGT GTT TGG AAT ACT AAA TGG AAA TCA GAT 2573Leu Phe Val Asp Ala Ala Ser Val Trp Asn Thr Lys Trp Lys Ser Asp 715 720725 AAA AAT GGA TTA GAG AGC AAG GTC TTG AAA GAC TTA CCT GAT TAT GGC 2621Lys Asn Gly Leu Glu Ser Lys Val Leu Lys Asp Leu Pro Asp Tyr Gly 730 735740 AAA TCA AGC CGT ATT CGC GCC TCT ACA GGT GTC GGA TTC CAA TGG CAA 2669Lys Ser Ser Arg Ile Arg Ala Ser Thr Gly Val Gly Phe Gln Trp Gln 745 750755 760 TCT CCT ATT GGA CCA TTG GTA TTT TCT TAT GCT AAA CCA ATT AAA AAA2717 Ser Pro Ile Gly Pro Leu Val Phe Ser Tyr Ala Lys Pro Ile Lys Lys 765770 775 TAT GAA AAT GAT GAT GTC GAA CAG TTC CAA TTT AGT ATT GGG GGC TCT2765 Tyr Glu Asn Asp Asp Val Glu Gln Phe Gln Phe Ser Ile Gly Gly Ser 780785 790 TTC TAATAAATTG AACTTTTTTC GTCATCAGAA CTCAAAAACG ACGTTCTCTG 2818Phe CCTAATTGAA TTGGGCAGAG AAAATATTAA ACCCATCATT TAATTAAGGA TATTTATCAA2878 ATGAAAAACA TCGCAAAAGT AACCGCACTT GCTTTAGGTT TTGCACTTGC TTCAGGCTAT2938 GCTTCCGCTG AAGAAAAAAT TGCTTTCATT AATGCAGGTT ATATTTTTCA A 2989 793amino acids amino acid linear protein not provided 10 Met Lys Lys LeuLeu Ile Ala Ser Leu Leu Phe Gly Ala Thr Thr Thr 1 5 10 15 Val Phe AlaAla Pro Phe Val Pro Lys Asp Ile Arg Val Asp Gly Val 20 25 30 Gln Gly AspLeu Glu Gln Gln Ile Arg Ala Ser Leu Pro Val Arg Ala 35 40 45 Gly Gln ArgVal Thr Asp Asn Asp Val Ala Asn Ile Val Arg Ser Leu 50 55 60 Phe Val SerGly Arg Phe Asp Asp Val Lys Ala His Gln Glu Gly Asp 65 70 75 80 Val LeuVal Val Ser Val Val Ala Lys Ser Ile Ile Ser Asp Val Lys 85 90 95 Ile LysGly Asn Ser Val Ile Pro Thr Glu Ala Leu Lys Gln Asn Leu 100 105 110 AspAla Asn Gly Phe Lys Val Gly Asp Val Leu Ile Arg Glu Lys Leu 115 120 125Asn Glu Phe Ala Lys Ser Val Lys Glu His Tyr Ala Ser Val Gly Arg 130 135140 Tyr Asn Ala Thr Val Glu Pro Ile Val Asn Thr Leu Pro Asn Asn Arg 145150 155 160 Ala Glu Ile Leu Ile Gln Ile Asn Glu Asp Asp Lys Ala Lys LeuAla 165 170 175 Ser Leu Thr Phe Lys Gly Asn Glu Ser Val Ser Ser Ser ThrLeu Gln 180 185 190 Glu Gln Met Glu Leu Gln Pro Asp Ser Trp Trp Lys LeuTrp Gly Asn 195 200 205 Lys Phe Glu Gly Ala Gln Phe Glu Lys Asp Leu GlnAla Ile Arg Asp 210 215 220 Tyr Tyr Leu Asn Asn Gly Tyr Ala Lys Ala GlnIle Thr Lys Thr Asp 225 230 235 240 Val Gln Leu Asn Asp Glu Lys Thr LysVal Asn Val Thr Ile Asp Val 245 250 255 Asn Glu Gly Leu Gln Tyr Asp LeuArg Ser Ala Arg Ile Ile Gly Asn 260 265 270 Leu Gly Gly Met Ser Ala GluLeu Glu Pro Leu Leu Ser Ala Leu His 275 280 285 Leu Asn Asp Thr Phe ArgArg Ser Asp Ile Ala Asp Val Glu Asn Ala 290 295 300 Ile Lys Ala Lys LeuGly Glu Arg Gly Tyr Gly Asn Thr Thr Val Asn 305 310 315 320 Ser Val ProAsp Phe Asp Asp Ala Asn Lys Thr Leu Ala Ile Thr Phe 325 330 335 Val ValAsp Ala Gly Arg Arg Leu Thr Val Arg Gln Leu Arg Phe Glu 340 345 350 GlyAsn Thr Val Ser Ala Asp Ser Thr Leu Arg Gln Glu Met Arg Gln 355 360 365Gln Glu Gly Thr Trp Tyr Asn Ser Gln Leu Val Glu Leu Gly Lys Ile 370 375380 Arg Leu Asp Arg Thr Gly Phe Phe Glu Thr Val Glu Asn Arg Ile Asp 385390 395 400 Pro Ile Asn Gly Ser Asn Asp Glu Val Asp Val Val Tyr Lys ValLys 405 410 415 Glu Arg Asn Thr Gly Ser Ile Asn Phe Gly Ile Gly Tyr GlyThr Glu 420 425 430 Ser Gly Ile Ser Tyr Gln Thr Ser Ile Lys Gln Asp AsnPhe Leu Gly 435 440 445 Thr Gly Ala Ala Val Ser Ile Ala Gly Thr Lys AsnAsp Tyr Gly Thr 450 455 460 Ser Val Asn Leu Gly Tyr Thr Glu Pro Tyr PheThr Lys Asp Gly Val 465 470 475 480 Ser Leu Gly Gly Asn Ile Phe Phe GluAsn Tyr Asp Asn Ser Lys Ser 485 490 495 Asp Thr Ser Ser Asn Tyr Lys ArgThr Thr Tyr Gly Ser Asn Val Thr 500 505 510 Leu Gly Phe Pro Val Asn GluAsn Asn Ser Tyr Tyr Val Gly Leu Gly 515 520 525 His Thr Tyr Asn Lys IleSer Asn Phe Ala Leu Glu Tyr Asn Arg Asn 530 535 540 Leu Tyr Ile Gln SerMet Lys Phe Lys Gly Asn Gly Ile Lys Thr Asn 545 550 555 560 Asp Phe AspPhe Ser Phe Gly Trp Asn Tyr Asn Ser Leu Asn Arg Gly 565 570 575 Tyr PhePro Thr Lys Gly Val Lys Ala Ser Leu Gly Gly Arg Val Thr 580 585 590 IlePro Gly Ser Asp Asn Lys Tyr Tyr Lys Leu Ser Ala Asp Val Gln 595 600 605Gly Phe Tyr Pro Leu Asp Arg Asp His Arg Trp Val Val Ser Ala Lys 610 615620 Ala Ser Ala Gly Tyr Ala Asn Gly Phe Gly Asn Lys Arg Leu Pro Phe 625630 635 640 Tyr Gln Thr Tyr Thr Ala Gly Gly Ile Gly Ser Leu Arg Gly PheAla 645 650 655 Tyr Gly Ser Ile Gly Pro Asn Ala Ile Tyr Ala Glu His GlyAsn Gly 660 665 670 Thr Phe Asn Lys Ile Ser Ser Asp Val Ile Gly Gly AsnAla Ile Thr 675 680 685 Thr Ala Ser Ala Glu Leu Ile Val Pro Thr Pro PheVal Ser Asp Lys 690 695 700 Ser Gln Asn Thr Val Arg Thr Ser Leu Phe ValAsp Ala Ala Ser Val 705 710 715 720 Trp Asn Thr Lys Trp Lys Ser Asp LysAsn Gly Leu Glu Ser Lys Val 725 730 735 Leu Lys Asp Leu Pro Asp Tyr GlyLys Ser Ser Arg Ile Arg Ala Ser 740 745 750 Thr Gly Val Gly Phe Gln TrpGln Ser Pro Ile Gly Pro Leu Val Phe 755 760 765 Ser Tyr Ala Lys Pro IleLys Lys Tyr Glu Asn Asp Asp Val Glu Gln 770 775 780 Phe Gln Phe Ser IleGly Gly Ser Phe 785 790 6 amino acids amino acid single linear notprovided 11 Ala Pro Phe Val Ala Lys 1 5 23 amino acids amino acid singlelinear not provided 12 Ser Leu Phe Val Ser Gly Arg Phe Asp Asp Val LysAla His Gln Glu 1 5 10 15 Gly Asp Val Leu Val Val Ser 20 9 amino acidsamino acid single linear not provided 13 Met Ser Pro Ile Leu Gly Tyr TrpLys 1 5 30 amino acids amino acid single linear not provided 14 Ala ProPhe Val Ala Lys Asp Ile Arg Val Asp Gly Val Gln Gly Asp 1 5 10 15 LeuGlu Gln Gln Ile Arg Ala Ser Leu Pro Val Arg Ala Gly 20 25 30 30 aminoacids amino acid single linear not provided 15 Pro Val Arg Ala Gly GlnArg Val Thr Asp Asn Asp Val Ala Met Ile 1 5 10 15 Val Arg Ser Leu PheVal Ser Gly Arg Phe Asp Asp Val Lys 20 25 30 32 amino acids amino acidsingle linear not provided 16 Gly Arg Phe Asp Asp Val Lys Ala His GlnGlu Gly Asp Val Leu Val 1 5 10 15 Val Ser Val Val Ala Lys Ser Ile IleSer Asp Val Lys Ile Lys Gly 20 25 30 30 amino acids amino acid singlelinear not provided 17 Ser Asp Val Lys Ile Lys Gly Asn Ser Val Ile ProThr Glu Ala Leu 1 5 10 15 Lys Gln Asn Leu Asp Ala Asn Gly Phe Lys ValGly Asp Val 20 25 30 30 amino acids amino acid single linear notprovided 18 Ala Asn Gly Phe Lys Val Gly Asp Val Leu Ile Arg Glu Lys LeuAsn 1 5 10 15 Glu Phe Ala Lys Ser Val Lys Glu His Tyr Ala Ser Val Gly 2025 30 30 amino acids amino acid single linear not provided 19 Val LysGlu His Tyr Ala Ser Val Gly Arg Tyr Asn Ala Thr Val Glu 1 5 10 15 ProIle Val Asn Thr Leu Pro Asn Asn Arg Ala Glu Ile Leu 20 25 30 31 aminoacids amino acid single linear not provided 20 Pro Asn Asn Arg Ala GluIle Leu Ile Gln Ile Asn Glu Asp Asp Lys 1 5 10 15 Ala Lys Leu Ala SerLeu Thr Phe Lys Gly Asn Glu Ser Val Ser 20 25 30 31 amino acids aminoacid single linear not provided 21 Phe Lys Gly Asn Glu Ser Val Ser SerSer Thr Leu Gln Glu Gln Met 1 5 10 15 Glu Leu Gln Pro Asp Ser Trp TrpLys Lys Leu Trp Gly Asn Lys 20 25 30 30 amino acids amino acid singlelinear not provided 22 Pro Asp Ser Trp Trp Lys Leu Trp Gly Asn Lys PheGlu Gly Ala Gln 1 5 10 15 Phe Glu Lys Asp Leu Gln Ser Ile Arg Asp TyrTyr Leu Asn 20 25 30 31 amino acids amino acid single linear notprovided 23 Leu Gln Ser Ile Arg Asp Tyr Tyr Leu Asn Asn Gly Tyr Ala LysAla 1 5 10 15 Gln Ile Thr Lys Thr Asp Val Gln Leu Asn Asp Glu Lys ThrLys 20 25 30 30 amino acids amino acid single linear not provided 24 ValGln Leu Asn Asp Glu Lys Thr Lys Val Asn Val Thr Ile Asp Val 1 5 10 15Asn Glu Gly Leu Gln Tyr Asp Leu Arg Ser Ala Arg Ile Ile 20 25 30 30amino acids amino acid single linear not provided 25 Tyr Asp Leu Arg SerAla Arg Ile Ile Gly Asn Leu Gly Gly Met Ser 1 5 10 15 Ala Glu Leu GluPro Leu Leu Ser Ala Leu His Leu Asn Asp 20 25 30 31 amino acids aminoacid single linear not provided 26 Pro Leu Leu Ser Ala Leu His Leu AsnAsp Thr Phe Arg Arg Ser Asp 1 5 10 15 Ile Ala Asp Val Glu Asn Ala IleLys Ala Lys Leu Gly Glu Arg 20 25 30 30 amino acids amino acid singlelinear not provided 27 Ala Ile Lys Ala Lys Leu Gly Glu Arg Gly Tyr GlySer Ala Thr Val 1 5 10 15 Asn Ser Val Pro Asp Phe Asp Asp Ala Asn LysThr Leu Ala 20 25 30 30 amino acids amino acid single linear notprovided 28 Phe Asp Asp Ala Asn Lys Thr Leu Ala Ile Thr Leu Val Val AspAla 1 5 10 15 Gly Arg Arg Leu Thr Val Arg Gln Leu Arg Phe Glu Gly Asn 2025 30 30 amino acids amino acid single linear not provided 29 Val ArgGln Leu Arg Phe Glu Gly Asn Thr Val Ser Ala Asp Ser Thr 1 5 10 15 LeuArg Gln Glu Met Arg Gln Gln Glu Gly Thr Trp Tyr Asn 20 25 30 30 aminoacids amino acid single linear not provided 30 Arg Gln Gln Glu Gly ThrTrp Tyr Asn Ser Gln Leu Val Glu Leu Gly 1 5 10 15 Lys Ile Arg Leu AspArg Thr Gly Phe Phe Glu Thr Val Glu 20 25 30 27 amino acids amino acidsingle linear not provided 31 Gly Phe Phe Glu Thr Val Glu Asn Arg IleAsp Pro Ile Asn Gly Ser 1 5 10 15 Asn Asp Glu Val Asp Val Val Tyr LysVal Lys 20 25 26 amino acids amino acid single linear not provided 32Asp Val Val Tyr Lys Val Lys Glu Arg Asn Thr Gly Ser Ile Asn Phe 1 5 1015 Gly Ile Gly Tyr Gly Thr Glu Ser Gly Ile 20 25 26 amino acids aminoacid single linear not provided 33 Gly Thr Glu Ser Gly Ile Ser Tyr GlnAla Ser Val Lys Gln Asp Asn 1 5 10 15 Phe Leu Gly Thr Gly Ala Ala ValSer Ile 20 25 28 amino acids amino acid single linear not provided 34Gly Ala Ala Val Ser Ile Ala Gly Thr Lys Asn Asp Tyr Gly Thr Ser 1 5 1015 Val Asn Leu Gly Tyr Thr Glu Pro Tyr Phe Thr Lys 20 25 27 amino acidsamino acid single linear not provided 35 Thr Glu Pro Tyr Phe Thr Lys AspGly Val Ser Leu Gly Gly Asn Val 1 5 10 15 Phe Phe Glu Asn Tyr Asp AsnSer Lys Ser Asp 20 25 26 amino acids amino acid single linear notprovided 36 Tyr Asp Asn Ser Lys Ser Asp Thr Ser Ser Asn Tyr Lys Arg ThrThr 1 5 10 15 Tyr Gly Ser Asn Val Thr Leu Gly Phe Pro 20 25 27 aminoacids amino acid single linear not provided 37 Val Thr Leu Gly Phe ProVal Asn Glu Asn Asn Ser Tyr Tyr Val Gly 1 5 10 15 Leu Gly His Thr TyrAsn Lys Ile Ser Asn Phe 20 25 28 amino acids amino acid single linearnot provided 38 Tyr Asn Lys Ile Ser Asn Phe Ala Leu Glu Tyr Asn Arg AsnLeu Tyr 1 5 10 15 Ile Gln Ser Met Lys Phe Lys Gly Asn Gly Ile Lys 20 2529 amino acids amino acid single linear not provided 39 Lys Gly Asn GlyIle Lys Thr Asn Asp Phe Asp Phe Ser Phe Gly Trp 1 5 10 15 Asn Tyr AsnSer Leu Asn Arg Gly Tyr Phe Pro Thr Lys 20 25 27 amino acids amino acidsingle linear not provided 40 Gly Tyr Phe Pro Thr Lys Gly Val Lys AlaSer Leu Gly Gly Arg Val 1 5 10 15 Thr Ile Pro Gly Ser Asp Asn Lys TyrTyr Lys 20 25 29 amino acids amino acid single linear not provided 41Ser Asp Asn Lys Tyr Tyr Lys Leu Ser Ala Asp Val Gln Gly Phe Tyr 1 5 1015 Pro Leu Asp Arg Asp His Leu Trp Val Val Ser Ala Lys 20 25 28 aminoacids amino acid single linear not provided 42 Leu Trp Val Val Ser AlaLys Ala Ser Ala Gly Tyr Ala Asn Gly Phe 1 5 10 15 Gly Asn Lys Arg LeuPro Phe Tyr Gln Thr Tyr Thr 20 25 26 amino acids amino acid singlelinear not provided 43 Phe Tyr Gln Thr Tyr Thr Ala Gly Gly Ile Gly SerLeu Arg Gly Phe 1 5 10 15 Ala Tyr Gly Ser Ile Gly Pro Asn Ala Ile 20 2527 amino acids amino acid single linear not provided 44 Gly Pro Asn AlaIle Tyr Ala Glu Tyr Gly Asn Gly Ser Gly Thr Gly 1 5 10 15 Thr Phe LysLys Ile Ser Ser Asp Val Ile Gly 20 25 29 amino acids amino acid singlelinear not provided 45 Lys Ile Ser Ser Asp Val Ile Gly Gly Asn Ala IleAla Thr Ala Ser 1 5 10 15 Ala Glu Leu Ile Val Pro Thr Pro Phe Val SerAsp Lys 20 25 27 amino acids amino acid single linear not provided 46Phe Val Ser Asp Lys Ser Gln Asn Thr Val Arg Thr Ser Leu Phe Val 1 5 1015 Asp Ala Ala Ser Val Trp Asn Thr Lys Trp Lys 20 25 26 amino acidsamino acid single linear not provided 47 Val Trp Asn Thr Lys Trp Lys SerAsp Lys Asn Gly Leu Glu Ser Asp 1 5 10 15 Val Leu Lys Arg Leu Pro AspTyr Gly Lys 20 25 27 amino acids amino acid single linear not provided48 Leu Pro Asp Tyr Gly Lys Ser Ser Arg Ile Arg Ala Ser Thr Gly Val 1 510 15 Gly Phe Gln Trp Gln Ser Pro Ile Gly Pro Leu 20 25 30 amino acidsamino acid single linear not provided 49 Gly Pro Leu Val Phe Ser Tyr AlaLys Pro Ile Lys Lys Tyr Glu Asn 1 5 10 15 Asp Asp Val Glu Gln Phe GlnPhe Ser Ile Gly Gly Ser Phe 20 25 30 89 base pairs nucleic acid singlelinear not provided 50 TATGGCACCT TTTGTGGCAA AAGATATTCG TGTGGATGGTGTTCAAGGTG ACTTAGAATC 60 AACAAACCGA GCAAGTTTAC CTGTTCGTG 89 92 basepairs nucleic acid single linear not provided 51 ACCGTGGAAA ACACCGTTTTCTATAAGCAC ACCTACCACA AGTTCCACTG AATCTTGGTT 60 GTTTAGGCTC GTTCAAATGGACAAGCACGG CC 92 23 base pairs nucleic acid single linear DNA (genomic)not provided 52 GGGGAATTCC AAAAGATGTT CGT 23 19 base pairs nucleic acidsingle linear not provided 53 CACGAATTCC CTGCAAATC 19 7 amino acidsamino acid single linear not provided 54 Met Ala Pro Phe Val Lys Asp 1 52987 base pairs nucleic acid single linear not provided 55 AAAAGGCATTGAAAAAACAG GACAGCTTTC CCTTTTAACC TTGAAAATAT TAGGGAAATT 60 ACTTACTGGCGATTTGTCAT TAAATAATTT AAGTGGGCCA ATTTCTATTG CAAAAGGTGC 120 TGGCGCATCAGCAAATATTG GATTGGTGTA TTTTTTAAGT TTTATGGCAT GATTAGTGTA 180 AATTTAGGGATTATGAATTT ATTTCCATTA CCAGTATTAG ATGGCGGTCA TTTAGTTTTT 240 TTAACAATGGAAGCTGTTAA AGGAAAACCT GTTTCTGAGC GGGTGCAAAG CATCTGTTAT 300 CGAATTGGCGCAGCACTGTT ATTAAGCTTA ACGGTGTTTG CATTATTTAA TGATTTTTTA 360 CGTCTATAATTTATATAGGA TACAATCGAT GAAAAAACTT CTAATCGCAA GTTTATTATT 420 CGGTACGACAACGACTGTGT TTGCCGCACC TTTTGTGGCA AAAGATATTC GTGTGGATGG 480 TGTTCAAGGTGACTTAGAAC AACAAATCCG AGCAAGTTTA CCTGTTCGTG CCGGTCAGCG 540 TGTGACTGACAATGATGTGG CTAATATTGT CCGCTCTTTA TTCGTAAGTG GTCGATTCGA 600 TGATGTGAAAGCGCATCAAG AAGGCGATGT GCTTGTTGTT AGCGTTGTGG CTAAATCGAT 660 CATTTCAGATGTTAAAATCA AAGGTAACTC TGTTATTCCC ACTGAAGCAC TTAAACAAAA 720 CTTAGATGCTAACGGGTTTA AAGTTGGCGA TGTTTTAATT CGAGAAAAAT TAAATGAATT 780 TGCCAAAAGTGTAAAAGAGC ACTATGCAAG TGTAGGTCGC TATAACGCAA CAGTTGAACC 840 TATTGTCAATACGCTACCAA ATAATCGCGC TGAAATTTTA ATTCAAATCA ATGAAGATGA 900 TAAAGCAAAATTGGCATCAT TAACTTTCAA GGGGAACGAA TCTGTTAGTA GCAGTACATT 960 ACAAGAACAAATGGAATTAC AACCTGATTC TTGGTGGAAA TTATGGGGAA ATAAATTTGA 1020 AGGTGCGCAATTCGAGAAAG ATTTGCAGTC AATTCGTGAT TATTATTTAA ATAATGGCTA 1080 TGCCAAAGCACAAATTACTA AAACGGATGT TCAGCTAAAT GATGAAAAAA CAAAAGTTAA 1140 TGTAACCATTGATGTAAATG AAGGTTTACA GTATGACCTT CGTAGTGCAC GCATTATAGG 1200 TAATCTGGGAGGTATGTCTG CCGAGCTTGA ACCTTTACTT TCAGCATTAC ATTTAAATGA 1260 TACTTTCCGCCGTAGTGATA TTGCAGATGT AGAAAATGCA ATTAAAGCAA AACTTGGAGA 1320 ACGCGGTTACGGTAGCGCAA CGGTAAATTC AGTACCTGAT TTTGATGATG CAAATAAAAC 1380 ATTAGCGATAACCCTTGTTG TTGATGCTGG ACGACGTTTA ACTGTTCGCC AACTTCGCTT 1440 TGAAGGAAATACCGTTTCTG CTGATAGCAC TTTACGTCAG GAAATGCGCC AACAAGAAGG 1500 AACTTGGTATAATTCACAAT TAGTTGAGTT AGGAAAAATT CGCTTAGATC GTACAGGTTT 1560 CTTCGAAACAGTCGAAAACC GAATTGATCC TATCAATGGT AGTAATGATG AAGTGGATGT 1620 CGTATATAAAGTCAAAGAAC GTAACACGGG TAGTATCAAC TTTGGTATTG GTTACGGTAC 1680 AGAGAGTGGTATTAGTTATC AAGCAAGTGT TAAACAAGAT AATTTCTTGG GAACAGGGGC 1740 GGCAGTAAGTATAGCTGGTA CGAAAAATGA TTATGGTACG AGTGTCAATT TGGGTTATAC 1800 CGAGCCCTATTTTACTAAAG ATGGTGTAAG TCTTGGTGGA AATGTTTTCT TTGAAAACTA 1860 CGATAACTCTAAAAGTGATA CATCCTCTAA CTATAAGCGT ACGACTTACG GAAGTAATGT 1920 TACTTTAGGTTTCCCTGTAA ATGAAAATAA CTCCTATTAT GTAGGATTAG GTCATACCTA 1980 TAATAAAATTAGTAACTTTG CTCTAGAATA TAACCGTAAT TTATATATTC AATCAATGAA 2040 ATTTAAAGGTAATGGCATTA AAACAAATGA CTTTGATTTT TCTTTTGGTT GGAACTATAA 2100 CAGCCTTAATAGAGGCTATT TCCCAACTAA AGGGGTTAAA GCAAGTCTTG GTGGACGAGT 2160 TACTATTCCAGGTTCTGATA ACAAATACTA CAAACTAAGT GCAGATGTAC AGGGTTTCTA 2220 CCCATTAGACAGAGATCACC TCTGGGTTGT ATCTGCAAAA GCATCTGCAG GATATGCAAA 2280 TGGTTTTGGAAACAAGCGTT TACCGTTCTA TCAAACTTAT ACAGCGGGTG GCATCGGTTC 2340 ATTACGTGGTTTTGCTTATG GTAGTATTGG ACCTAACGCA ATTTATGCCG AATATGGTAA 2400 TGGTAGTGGTACTGGTACTT TTAAGAAGAT AAGTTCTGAT GTGATTGGTG GTAATGCAAT 2460 CGCTACAGCTAGCGCAGAGT TAATTGTGCC AACTCCATTT GTGAGCGATA AGAGCCAAAA 2520 TACGGTCCGAACCTCCTTAT TTGTTGATGC GGCAAGTGTT TGGAATACTA AATGGAAATC 2580 AGATAAAAATGGATTAGAGA GCGATGTATT AAAAAGATTG CCTGATTATG GCAAATCAAG 2640 CCGTATTCGCGCCTCTACAG GTGTCGGATT CCAATGGCAA TCTCCTATTG GGCCATTGGT 2700 ATTCTCTTATGCCAAACCAA TTAAAAAATA TGAAAATGAT GATGTCGAAC AGTTCCAATT 2760 TAGTATTGGAGGTTCTTTCT AATAAATTGA ACTTTTTTCT TCATCAGAAC TCAAAAACAA 2820 CGTTCTCTGCCTAATTTAAT TGGGCAGAGA AAATATTAAA CCCATCATTT AATTAAGGAT 2880 ATTTATCAAATGAAAAACAT CGCAAAAGTA ACCGCACTTG CTTTAGGTAT TGCACTTGCT 2940 TCAGGCTATGCTTCCGCTGA AGAAAAAATT GCTTTCATTA ATGCAGT 2987

What we claim is:
 1. An immunogenic peptide consisting of an amnio acidsequence which is that of a portion only of the amino acid sequence of apurified and isolated D15 outer membrane protein having a molecularweight of about 80 kDa, said peptide being selected from the groupconsisting of peptides consisting of SEQ ID NOS: 14-38, 40, 41 and45-49.
 2. An immunogenic composition, comprising at least oneimmunologically-active component which is selected from the groupconsisting of: (a) a purified and isolated Haemophilus influenzae D15outer membrane protein having a molecular weight as determined bySDS-PAGE of about 80 kDA and selected from the group consisting of: (i)an amino acid sequence consisting of SEQ ID NOS: 2, 4, 6, 8 or 10; (ii)an amino acid sequence encoded by DNA sequence consisting of SEQ ID NOS:1, 3, 5, 7 or 9; and (iii) an amino acid sequence encoded by a DNAsequence consisting of a consensus DNA sequence consisting of SEQ ID NO:55; (b) an immunogenic synthetic peptide consisting of an amino acidsequence which is that of a portion only of the amino acid sequence of apurified and isolated D15 outer membrane protein, said peptide beingselected from the group consisting of peptides consisting of SEQ ID Nos.14-36, 40, 41 and 45-49; and (c) a chimeric molecule comprising theprotein in (a) or the peptide in (b) linked to a foreign polypeptide orprotein or to a polysaccharide; and a physiologically-acceptable carriertherefor.
 3. The immunogenic composition of claim 2 formulated as avaccine for in vivo administration to protect against diseases caused byHaemophilus.
 4. The immunogenic composition of claim 3 formulated as amicroparticle preparation, capsule preparation or liposome preparation.5. The immunogenic composition of claim 3 in combination with atargeting molecule for delivery to specific cells of the immune systemor to mucosal surfaces.
 6. A method of inducing protection againstdisease caused by Haemophilus, comprising administering to a subject aneffective amount of the immunogenic composition of claim 2 to provideprotection against Haemophilus disease.
 7. A chimeric molecule,comprising: (a) a purified and isolated D15 outer membrane proteinhaving a molecular weight of about 80 kDa, wherein said outer membraneprotein is selected from the group consisting of: (i) an amino acidsequence consisting of SEQ ID NOS: 2, 4, 6, 8 or 10; (ii) an amino acidsequence encoded by a DNA sequence consisting of SEQ ID NOS: 1, 3, 5, 7or 9; and (iii) an amino acid sequence encoded by a DNA sequenceconsisting of a consensus DNA sequence consisting of SEQ ID NO: 55 and(b) an immunogenic synthetic peptide consisting of an amino acidsequence which is that of a portion only of the amino acid sequence of apurified and isolated D15 outer membrane protein having a molecularweight of about 80 kDa, said peptide being selected from the groupconsisting of peptides consisting of SEQ ID NOS: 14-38, 40, 41 and 45-49linked to a foreign polypeptide or protein or to a polysaccharide. 8.The chimeric molecule of claim 7 wherein said D15 outer membrane proteinis linked to a foreign polypeptide or protein which comprises a surfaceprotein or peptide from a pathogenic bacteria.
 9. The chimeric moleculeof claim 8 wherein said foreign polypeptide or protein comprises a P1,P2, or P6 outer membrane of H. influenzae.
 10. The chimeric molecule ofclaim 7 wherein said D15 outer membrane protein is linked to apolysaccharide which comprises a PRP molecule from H. influenzae.
 11. Apurified and isolated Haemophilus influenzae D15 outer membrane proteinhaving a molecular weight as determined by SDS-PAGE of about 80 kDa andconsisting of an amino acid sequence which is selected from the groupconsisting of: (a) an amino acid sequence consisting of SEQ ID NOS: 2,4, 6, 8 or 10; (b) an amino acid sequence encoded by a DNA sequenceconsisting of SEQ ID NOS: 1, 3, 5, 7 or 9; and (c) an amino acidsequence encoded by a DNA sequence consisting of a consensus DNAsequence consisting of SEQ ID NO:
 55. 12. The protein of claim 11wherein the Haemophilus influenzae is a Haemophilus influenzae type bstrain selected from the group consisting of Ca, MinnA and Eaganstrains.
 13. The protein of claim 11 wherein the Haemophilus influenzaeis a non-typeable Haemophilus influenzae strain selected from the groupconsisting of PAK12085 and SB33 strains.
 14. A purified and isolatedHaemophilus influenzae D15 outer membrane protein consisting of an aminoacid sequence which has SEQ ID NO: 8 or consisting of an amino acidsequence which is encoded by a nucleic acid molecule which has SEQ IDNO:7.